Radiosensitizing effects of silver nanoparticles targeting angiogenesis and matrix metalloproteinase signaling in triple negative breast cancer cell lines.

Introduction
Cancer remains a significant global threat due to its high incidence and mortality rates1,2. Among women, breast cancer is the most prevalent diagnosed malignancy, accounting for approximately 24.5% of all reported cancer cases3,4. Triple-negative breast cancer (TNBC), the most aggressive subtype, is defined by the absence of receptors (ER–/PR–/HER2–), making it difficult to treat. The aggressiveness of TNBC is primarily attributed to its enhanced angiogenic and metastatic potential, which supports rapid tumor growth and a strong tendency for metastasis5,6.
Radiation therapy (RT) remains a cornerstone in breast cancer treatment and employs high-energy photons to control local tumors by eradicating residual cancer cells7. RT damages DNA directly by inducing strand breaks or indirectly by generating reactive oxygen species (ROS), which cause oxidative damage to DNA8. However, conventional RT can also harm surrounding normal tissues, highlighting the need for precise radiation dose to minimize collateral damage9.
Several studies have demonstrated that RT upregulates matrix metalloproteinases (MMP‑2/‑9), enzymes responsible for cancer cell migration, invasion, and angiogenesis 10,11. These adverse effects hinder the therapeutic efficacy of RT, particularly in TNBC12, emphasizing the need for a radiosensitizer that not only enhances tumor cell eradication but also suppresses RT-induced pro-metastatic signaling13. Nanotechnology-based radiosensitizers have emerged as promising agents to overcome these limitations by improving tumor selectivity and minimizing off-target toxicity. Silver nanoparticles (Ag-NPs) have attracted much attention in cancer treatment due to their strong cytotoxic, pro-apoptotic, and anti-proliferative properties14. Ag-NPs induce ROS generation and cause DNA damage, resulting in apoptotic cell death15. Ag-NPs also downregulate MMP-2 and -916, facilitating cell migration and invasion17.
Angiogenesis is a key driver of tumor growth and metastasis, making it a critical target in cancer therapy. The vascular endothelial growth factor (VEGF) -A/ VEGF receptor (VEGFR2) is a major regulator of angiogenesis18; however, tumors can also employ VEGF-independent mechanisms to sustain vascularization19. In addition to classical angiogenesis, aggressive tumors, such as TNBC, can exploit alternative strategies, including vascular mimicry (VM), to form vessel-like structures independently of endothelial cells20. A subset of cancer stem-like cells drives VM by acquiring endothelial characteristics, including VEGFR2 and Tyrosine kinase with immunoglobulin-like and EGF-like domains 2 (Tie2) expression, which facilitates integration of tumor-derived channels with host vasculature and promotes metastatic spread. Therefore, targeting angiogenesis and VM, particularly through modulation of VEGFR2 and Tie2, may offer a more effective strategy to suppress tumor vascularization and metastasis in TNBC21,22. To model these processes in vitro, a tube formation assay that captures both endothelial angiogenesis and TNBC-mediated VM can provide a functional readout of tumor vascularization.
Given the radiosensitizing potential of Ag-NPs through induction of apoptosis and inhibition of proliferation23, we elucidated the impact of Ag-NPs alone and in combination with RT on angiogenesis and migration in TNBC. To explore the underlying mechanisms, we assessed the expression of VEGFR2 and Tie2, as well as MMP-2 and MMP-9, key mediators of vascularization and invasion. By targeting these interconnected pathways and functionally assessing tube formation, this study provides mechanistic insight into how Ag-NPs may enhance the therapeutic efficacy of RT while simultaneously disrupting tumor vascularization and VM-associated metastatic processes in TNBC.

Materials and methods

Cell culture and reagents
MDA-MB-231 and MDA-MB-468 cells, as TNBC models, were provided by the Pasteur Institute (Tehran, Iran). Both cells were grown in RPMI-1640 medium (Gibco, USA), containing 10% FBS (Gibco, USA) and 1% penicillin–streptomycin (Sigma, USA). The cells were maintained in a humidified atmosphere containing 5% CO2 at 37 °C. The Ag-NPs were prepared by US Research Nanomaterials Inc. and obtained from the Iranian Nanomaterial Pioneers Company (Mashhad City, Khorasan, Iran). Cells were grown to 70–80% confluency and then subjected to the designed treatment regimens. Cell culture plates and dishes were purchased from SPL Life Sciences (South Korea). Other reagents were provided by Gibco Co. (UK).

Ag-NPs preparation
Distilled water was used to disperse polyvinylpyrrolidone (PVP)-coated Ag-NP powder without the addition of surfactants. As described in a previous publication by this group, the size of Ag-NPs is 50–80 nm with a purity of 99%24. The commercial supplier reported the scanning electron microscopy (SEM) analysis, zeta potential, dynamic light scattering (DLS) analysis, and size distribution, as described in our previous work24.

Radiation
The cell culture flasks were irradiated using an Elekta linear accelerator (Elekta Solutions AB, Sweden). The flasks were positioned between water-equivalent slab phantoms at the source to a surface distance of 100 cm with a field size of 20 × 20 cm2. The cells received a total dose of 4 or 8 Gray from 6 MV photons at a dose rate of 300 cGy/min.

Assessment of cell toxicity and cell viability
To investigate the in vitro cytotoxic effects of Ag-NPs on MDA-MB-468 cells and MCF-10A cells, an MTT assay was performed. The IC50 value for MDA-MB-231 cells, as reported in our published study, was 3.99 ± 0.51 μg/ml25. For the current experiment, MDA-MB-468 and MCF-10A cells were seeded at a density of 3 × 103 per well in a 96-well plate containing 200 μl of complete culture medium. Following a 24 h incubation period, cells were treated with different concentrations of Ag-NPs (ranging from 0.1 to 15.5 μg/ml). After 48 h of incubation under standard conditions (humid atmosphere, 5% CO2), 20 μl of MTT solution (5 mg/ml) was added to each well.
The plate was incubated for 4 h to facilitate the formation of formazan crystals. After incubation, the supernatants were carefully removed, and the crystals were solubilized using dimethyl sulfoxide (DMSO). The absorbance was measured at 570 nm using a microplate reader (BioTek Instruments, Inc., USA). Untreated cells served as negative controls. IC50 was obtained from the dose–response curve, and statistical analysis was conducted using GraphPad Prism software. All experiments were performed in triplicate across three independent runs, and data are presented as mean ± standard deviation.
To further assess cell viability following irradiation, a trypan blue exclusion dye assay was performed. MDA-MB-231 and MDA-MB-468 cells were cultured in T25 flasks for 24 h and subjected to treatment groups: untreated control, Ag-NPs alone, RT alone, or combination therapy. For combination treatment, MDA-MB-231 and MDA-MB-468 cells were exposed to the determined concentrations of Ag-NPs and incubated for 2 h prior to irradiation at doses of 4 and 8 Gray. RT doses of 4 and 8 Gray were applied, following protocols adapted from prior studies26,27 . After 48 h, cell viability was evaluated under a microscope using Trypan Blue exclusion dye28. Each condition was tested in triplicate across three independent experiments. The combination index (CI) for Ag-NPs and RT was determined using the Chou-Talalay method. The combination index (CI) for Ag-NPs and RT was calculated using the Chou-Talalay method, which quantitatively defines drug interactions: a CI < 1 indicates synergism, CI > 1 indicates antagonism, and CI = 1 denotes an additive effect.

Quantitation of apoptosis using the Annexin V/PI staining assay
The apoptotic cell population was identified using an apoptosis detection kit in a flow cytometry assay (Cat No: ED7044; EXBIO). MDA-MB-231 and MDA-MB-468 cells were seeded at 1 × 105 cells per well in 6-well plates and incubated overnight. Cells were then exposed to either Ag-NPs alone, irradiation alone, or a combination of Ag-NPs and irradiation for 48 h. The cells were collected from the control group (untreated cells), cells treated with Ag-NPs alone, irradiated cells, and cells treated with Ag-NPs plus irradiation for 48 h. In the following, the cells were harvested and washed with phosphate-buffered saline (PBS), then resuspended in binding buffer for 20 min at 4 °C. Subsequently, the cells were incubated with 5 µL of FITC-conjugated Annexin V in 100 µL of binding buffer in the dark for 15 min at room temperature. After a final wash with binding buffer, cells were stained with 5 µL of propidium iodide (PI) in 100 µL of binding buffer. Apoptotic cells were determined using a FACSCalibur (BD Bioscience), and data were analyzed using the FlowJo software version X.V-10. All experiments were performed in triplicate29,30.

Flow Cytometry for evaluation of ROS production
Intracellular ROS levels were assessed using the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma Chemical Co., St. Louis, MO, USA). DCFH-DA is a non-fluorescent, oxidation-sensitive probe that, after cellular uptake, is deacetylated and subsequently oxidized by ROS to form the highly fluorescent compound 2′,7′-dichlorofluorescein (DCF), which emits green fluorescence. MDA-MB-231 and MDA-MB-468 were cultured, treated, and harvested as described in previous experiments. The cells were incubated with 500 μl of DCFH-DA (10 μM PBS) at 37 °C in a CO2 incubator for 2 h. Following incubation, cells from all experimental groups were detached, washed 3 times with PBS, and resuspended in 500 μl PBS. Fluorescence intensity, indicative of ROS generation, was measured using a FACSCalibur flow cytometer (BD Biosciences, USA).

Determination of VEGFR2 and Tie2 expression levels using real-time PCR
Quantitative real-time PCR (qRT-PCR) was used to determine the mRNA expression of angiogenesis-associated genes VEGFR2 and Tie2 in MDA-MB-231 and MDA-MB-468 cells following treatment. Total RNA was extracted using a Thermo Scientific kit (K0731) according to the manufacturer’s instructions. RNA purity and concentration were determined in each group using a Nanodrop (ND-1000, Wilmington, DE, USA). For cDNA synthesis, 500 ng of total RNA was reverse-transcribed using a method described in a prior publication31. Gene expression was quantified using SYBR Green-based qPCR. β-Actin served as the internal control, and all collected data were normalized based on β-Actin expression. The primer sets utilized are listed in Table 129. All reactions were performed in triplicate, and relative gene expression was calculated using the 2−ΔΔCt method, which remains the gold standard for comparative quantification in gene expression studies.

Determination of MMP2 and MMP9 protein levels using Western blotting analysis
Protein extraction was performed for all experimental groups, including untreated control cells, cells treated with Ag-NPs, cells exposed to RT alone, and cells treated with a combination of RT and Ag-NPs. Harvested cells were rinsed with cold PBS and lysed using RIPA buffer at 4 °C for 30 min. Lysates were centrifuged at 12,000 × g for 15 min at 4 °C, and the supernatants were collected for protein quantification using the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA). Fifty micrograms of each lysate were subjected to electrophoresis on SDS-PAGE gels followed by transfer to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in TBS-T buffer containing 5% non-fat dry milk for 1 h at room temperature to prevent non-specific binding. Following a 60-min incubation at 25 °C, the membranes were incubated with primary antibodies (1:1000) against the desired proteins, including pro-MMP-2, cleaved-MMP-2 (Santa Cruz Biotechnology, sc-6838, USA), MMP-9 (Santa Cruz Biotechnology, sc-10737, USA), and β-actin (Santa Cruz Biotechnology, sc-517582, USA) overnight at 4 °C.
After washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000) from Santa Cruz (mouse anti-goat IgG-HRP: sc-2354, and mouse anti-rabbit IgG-HRP; sc-23571:5000) for 1 h at room temperature to detect the binding of primary antibodies. Protein bands were developed by enhanced chemiluminescence (ECL) reagents (Roche, UK) and captured on X-ray film. The intensity of each protein was determined using ImageJ 1.6 and normalized to the corresponding β-actin control32,33.

Wound-healing assay to determine the effect of Ag-NPs on the cell migration
To determine whether Ag-NPs affect cell migration, a wound-healing assay was conducted. The cells were cultured in 6-well plates at a density of 4 × 105 cells per well to form a monolayer at 90% confluency for the assay. Then, a scratch was manually created at the center of each well using a sterile micropipette tip, followed by a wash with DPBS (Dulbecco’s Phosphate-Buffered Saline) to remove any cellular debris. Following this, the cells were exposed to the treatment regimen, and their migration was assessed at various time intervals (0, 24, and 48 h). The width of the cell-free area was monitored using an inverted microscope (KRÜSS, MBL-3200) at 10X magnification. Cell-free areas were quantified using ImageJ. The assay was repeated three times, and each sample was run in triplicate.

Tube formation assay to determine the effect of Ag-NPs on the cell angiogenesis
To investigate angiogenesis, an indirect co-culture system was established using MDA-MB-231 and HUVEC. Briefly, HUVECs (2 × 105) were seeded into the lower chamber of a 24-well Transwell system pre-coated with 150 μl of Matrigel (BD Biosciences) and allowed to adhere for 30 min. Subsequently, MDA-MB-231 cells (4 × 104) were seeded into the upper chamber. This two-chamber system separated by a porous membrane, allows pro-angiogenic factors secreted by MDA-MB-231 cells to diffuse toward the HUVECs, thereby stimulating angiogenic responses. After 6 h of incubation, HUVECs were evaluated for the formation of capillary-like tube structures. Images were acquired from three random fields per sample using an inverted microscope (Nikon TE2000-S) at 4 × magnification, and tube formation was quantified using ImageJ. To confirm endothelial tube formation, CD31 (PECAM-1) immunofluorescence staining was performed. CD31 is a well-established endothelial marker used to visualize and quantify angiogenic structures34. The number of junctions was measured to assess the extent of vessel-like structure formation.

Statistical analysis
All quantitative data were analyzed using GraphPad Prism software version 8. Group comparisons were performed using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test for comparison between groups. Results were expressed as mean ± standard deviation (SD), and statistical significance was defined as p < 0.05.

Results

Cytotoxic effects of Ag-NPs on MDA-MB-23, MDA-MB-468, and MCF-10A
To evaluate the potential of Ag-NPs to enhance the anticancer efficacy of RT, we assessed their cytotoxicity in two TNBC cell lines, MDA-MB-231 and MDA-MB-468, and the non-tumorigenic epithelial cell line MCF-10A (as a control). In our previous study25; the IC₅₀ value of Ag-NPs for MDA-MB-231 cells was determined to be 3.99 ± 0.51 µg/ml using the MTT assay.
Based on the obtained data, the IC50 values for Ag-NPs were determined to be 6.76 ± 1.3 µg/mL and 20.4 ± 1.9 µg/ml in MDA-MB-468 and MCF-10A cell lines, respectively. As shown, the IC50 value of Ag-NPs in MCF-10A cells is remarkably higher than that of TNBC cell lines (Fig. 1a). Therefore, the two TNBC cell lines were used to study combination therapy. To minimize cytotoxicity associated with higher concentrations, doses around or below the IC50 value were selected for the combination therapy with Ag-NPs and RT. Indeed, the MTT assay and Compusyne software were used to determine the optimal dose for the combination. Based on this finding, 2 and 4 µg/ml were selected for subsequent monotherapy and combination therapy experiments in the MDA-MB-231 cell line. 5 and 7 µg/ml were selected for combination therapy experiments in the MDA-MB-468 cell line. RT doses of 4 and 8 Gray were applied in accordance with established protocols26,27.
Table 2 presents the inhibitory effects, dose-reduction index (DRI), and CI values obtained by Compusyne software after treating MDA-MB-231 cells with various combination therapies. The results reveal that all combination groups exhibit a synergistic anticancer effect on MDA-MB-231 cancer cells. As defined, CI < 1 represents a synergistic effect. Among different combination therapies for MDA-MB-231 cells, the combination of 4 µg/ml Ag-NPs with 4 and 8 Gray showed the most synergistic effects, with combination CI values of 0.42 and 0.44, respectively. Therefore, we selected a combination therapy for further experiments, including a low dose of RT (4 Gray) combined with 4 µg/ml Ag-NPs. The Fa-CI plot of the selected combination therapy and the anticancer effects of each monotherapy and combination therapy in MDA-MB-231 cells are shown in Fig. 1b. As shown in the Fig. 1b, the cell viability percentage in combination therapy (4 µg/ml Ag-NPs and 4 Gray RT) reached 29.05 ± 4.78, from 49.45 ± 1.67 and 76.67 ± 2.36.in Ag-NP- and RT-treated groups, respectively (p < 0.001).
As shown in the Fig. 1b, the cell viability percentage in combination therapy (4 µg/ml Ag-NPs and 4 Gray RT) reached 29.05 ± 4.78, from 76.67 ± 2.36 and 49.45 ± 1.67 in the RT- and Ag-NP-treated groups, respectively (p < 0.001). Table 3 presents the inhibitory effects, DRI, and CI values derived from CompuSyn software following treatment of MDA-MB-468 cells with Ag-NPs and RT. All tested combinations demonstrated synergistic interactions, as indicated by CI values below 1 (0.53–0.73), confirming the enhanced anticancer efficacy of the combined treatment. A combination of 7 µg/ml Ag-NPs and 8 Gray radiation therapy resulted in the best CI value of 0.53. Therefore, this combination was selected for use in the following experiments. Figure 1c also shows the Fa-CI plot of selected combination therapy and the anticancer effect of each monotherapy and combination therapy in the MDA-MB-468 cell line. Treatment of MDA-MB-468 cells with a combination of 7 µg/ml Ag-NPs and 8 Gray RT significantly reduced cell viability to 23.30 ± 3.3% from 50.45 ± 3.75 and 69.22 ± 2.3%, respectively, in Ag-NP- and RT-treated cells. These findings are consistent with the results observed in MDA-MB-231 cells, reinforcing the reproducibility and reliability of Ag-NPs as a radiosensitizer across multiple TNBC models.

Ag-NPs enhanced radiation-induced apoptosis in MDA-MB-231 and MDA-MB-468 cells
To determine the pro-apoptotic effects of Ag-NPs, both as monotherapy and in combination with irradiation, we performed Annexin V/ PI double staining followed by flow cytometric analysis in two TNBC cell lines: MDA-MB-231 and MDA-MB-468. After 48 h of treatment, the cells were harvested and analyzed. Figure 2a depicts the contour diagram of Annexin V/ PI flow cytometry analysis of MDA-MB-231 cells. Compared to untreated groups, Ag-NPs alone induced a significant increase in apoptosis (43.24 ± 2.17%, p < 0.001), while RT alone resulted in 17.44 ± 1.93% apoptotic cells. Notably, the combination therapy markedly increased the population of apoptotic cells (59.64 ± 0.16) compared with either monotherapy (p < 0.001).
This experiment was carried out in MDA-MB-468 cells to validate the consistency of the apoptotic response in a second TNBC model (Fig. 2b). A similar trend was observed in MDA-MB-468 cells, as Ag-NPs and RT monotherapies induced apoptosis rates of 46.84 ± 5.6 and 28.45 ± 2.8, respectively (p < 0.001), compared to the control group. While the combination therapy markedly increases the apoptotic population to 75.98 ± 3.4, compared to each monotherapy (p < 0.001). The consistent enhancement of apoptosis across both cell lines underscores the potential of Ag-NPs as an effective radiosensitizer in TNBC therapy. The results from three independent experiments are depicted in a bar graph, while the results from one experiment are illustrated as a dot plot histogram.

Evaluation of ROS in MDA-MB-231 and MDA-MB-468 cells underwent exposure to monotherapies and combination therapy
Apoptosis is the preferred form of cell death in cancer cells, and ROS plays a key role in its induction35. To evaluate the impact of each treatment on intracellular ROS production following the completion of the treatment protocol, the ROS level was measured by flow cytometry analysis. As depicted in Fig. 3a, the level of ROS has notably increased in MDA-MB-231 cells exposed to the combination of Ag-NP and RT compared with each treatment alone (p < 0.001). However, a single treatment with RT and Ag-NPs induced ROS production as compared to the control group (p < 0.001). In MDA-MB-468 cells, the percentage of ROS was also significantly increased in the group that received the combination of Ag-NP and RT (Fig. 3b) as compared to cells treated with Ag-NP and RT alone (p < 0.01 and p < 0.001, respectively).

Combination of Ag-NPs with RT modulates VEGFR2 and Tie2 expression in MDA-MB-231and MDA-MB-468 cells
In this study, we investigated the impact of Ag-NPs, alone and in combination with RT, on the expression of the angiogenesis-related genes VEGFR2 and Tie2 in TNBC cell lines MDA-MB-231 and MDA-MB-468. These genes are central to classical angiogenesis and VM, a process by which tumor cells form vessel-like structures independent of endothelial cells. VEGFR2 facilitates endothelial-like behavior in tumor cells, promoting channel formation and metastatic dissemination, while Tie2, through its interaction with angiopoietins, contributes to vascular stabilization and supports trans differentiation into VM phenotypes33,36. Dysregulation of these pathways is closely associated with the aggressive and metastatic nature of TNBC.
Figure 4a shows that Ag-NPs significantly downregulated VEGFR2 and Tie2compared with untreated controls (p < 0.001 for VEGFR2 and p < 0.01 for Tie2). Notably, the combination of Ag-NPs with RT potently inhibited mRNA expression in the MDA-MB-231 cell line compared with each monotherapy group (p < 0.001 vs RT and p < 0.05 vs Ag-NPs).
A similar trend was observed in MDA-MB-468 cells, with Ag-NP monotherapy downregulating Tie2 and VEGFR2 expression levels (p < 0.001) compared to the control group. The combination therapy markedly downregulated VEGFR2 expression compared with RT and Ag-NPs alone (p < 0.001 and p < 0.05, respectively). Tie2 mRNA levels also significantly decreased in RT p < 0.001 and Ag-NPs alone p < 0.01 (Fig. 4b). These comparable results across both TNBC cell lines highlight the consistent inhibitory impact of Ag-NPs combined with RT on angiogenic signaling, supporting their potential utility in targeting vascular pathways in aggressive breast cancer.

A combination of Ag-NPs with RT altered the Level of MMP-2 and MMP-9
MMP-2 and MMP-9 play a pivotal role in the degradation of the extracellular matrix, which is a critical initial step in the complex process of breast cancer invasion and metastasis, making their examination essential for understanding tumor progression mechanisms in this study37. Although RT contributes to MMP activation, IR-produced ROS directly interacts with MMPs and oxidizes the critical sites needed for MMP activation. Therefore, we examined the effects of combination therapy on MMP2/9 expression compared to their corresponding monotherapies. Western blot analysis was used to determine whether monotherapies and combination therapy altered the expression levels of MMP-2 and MMP-9 in MDA-MB-231 cells (Fig. 5a–e). The results (Fig. 5b and e) revealed that both monotherapies (RT and Ag-NPs) significantly reduced the expression levels of MMP9 and MMP2-cleaved/pro-MMP2 compared to the control group (p < 0.001). Interestingly, the combination of Ag-NPs and RT intensified the reduction of MMP9 and MMP2-cleaved/pro-MMP2 compared with each monotherapy (p < 0.001).

A combination of Ag-NPs with RT inhibited the migration and angiogenesis of MDA-MB-231 Cells
Given the significant role of cell migration in cancer metastasis38, we conducted a wound-healing assay to investigate the impact of Ag-NPs, RT, and their combination on cell migration. The migration inhibitory effect was performed by measuring cell-free areas in MDA-MB-231 cells, and the inhibitory effect on migration was determined by measuring cell-free areas. As depicted in Fig. 6a, Ag-NPs significantly reduced cell migration, and combination therapy exerted a greater inhibitory effect than the control group at time 0 (untreated cells). Our findings revealed reduced migration in Ag-NP-treated cells, and this effect was potentiated by combination therapy.
As shown in Fig. 6b, the number of junctions formed during tube formation was markedly reduced following treatment. Compared with the control group, RT and Ag-NPs alone significantly reduced the number of junctions (p < 0.001), whereas the combination of RT and Ag-NPs exhibited the most pronounced inhibitory effect (p < 0.01 compared to RT alone). These findings indicate that the combination therapy strongly suppressed the angiogenic potential of MDA-MB-231 cells.
Representative microscopic images of wound healing assay at various time points (0, 24, and 48 h) and tube formation assay from all experimental groups were presented in Supplementary File; Sects. 2 and 3, respectively.

Discussion
TNBC is the most lethal subtype of breast cancer due to its aggressive clinical features, lack of therapeutic targets, and resistance to conventional treatments. Angiogenesis and metastasis are key drivers of TNBC progression, contributing to aggressive behavior and poor clinical outcomes. Given the reliance of TNBC on angiogenesis, targeting these pathways has become a critical focus for developing effective therapeutic strategies39. While chemotherapy and radiotherapy remain primary treatment modalities for TNBC patients, their efficacy is often limited by resistance and other factors. In this context, Ag-NPs have gained attention as promising radiosensitizer in various cancers, including TNBC40,41, where our previous study demonstrated their apoptotic and radiosensitizing effects27.
Nanoparticles, particularly those with high atomic numbers such as Ag-NPs, enhance the local effects of MV radiation by increasing secondary electron production through Compton scattering and photoelectric interactions. Secondary electrons produced by photon interactions with NPs can also initiate a cascade of further ionizations and secondary electron emissions. These cascades are particularly effective in generating multiple ionizations in the local environment, further enhancing the overall radiation damage in the tissue. These secondary electrons enhance ROS generation, which amplifies DNA and cellular damage, improving the radiation-induced cytotoxicity42,43.
In our study, the normal mammary epithelial cell line MCF-10A lacks oncogenic alterations, exhibits limited sensitivity to Ag-NPs, with cytotoxic effects observed only at higher concentrations. In TNBC cell lines, the observed higher IC₅₀ of Ag-NPs in MDA-MB-468 cells, compared to MDA-MB-231 cells, indicates that MDA-MB-468 cells are relatively less sensitive to Ag-NP-induced cytotoxicity. Similarly, the higher radiation dose required to achieve comparable effects in MDA-MB-468 cells suggests an intrinsic radioresistance in this cell line relative to MDA-MB-231, likely reflecting inherent biological and molecular variations.
Importantly, Ag-NPs alone and in combination with RT significantly induced apoptosis in MDA-MB-231 and MDA-MB-468 cells, correlating with elevated ROS levels. This supports ROS as a central mediator of apoptosis, likely through mitochondrial dysfunction, DNA damage, and activation of intrinsic apoptotic pathways. Consistent with previous reports, the combination of Ag-NPs with RT exhibited a synergistic effect in MDA-MB-231 breast cancer cells27. Moreover, the enhanced apoptotic effect of combination therapy was confirmed in MDA-MB-468 cell line. Apoptosis is an essential and desirable mechanism of cell death in cancer therapy. Ag-NPs inhibit cell growth by eliciting apoptosis in cancer cells44,45. Our previous report25 demonstrated that the provided Ag-NPs upregulated pro-apoptotic genes, including p53 and Bax, while downregulating the anti-apoptotic gene Bcl-2 in MDA-MB-231 cells, further supporting the efficacy of Ag-NPs in promoting apoptosis in cancer cells.
Angiogenesis, the formation of new blood vessels from pre-existing capillaries, is essential for tumor progression, supplying oxygen and nutrients for rapid growth. During this process, endothelial cells proliferate and migrate to form nascent networks for supplying oxygen and nutrients needed for growth46. A high level of angiogenesis is correlated with a decreased survival rate in breast cancer patients39. VEGFR-2, a receptor on endothelial cells, is activated by VEGF, a key driver of angiogenesis. Upon binding of VEGF to VEGFR-2, it triggers signaling that promotes endothelial cell proliferation, migration, and angiogenesis, a process essential for supplying oxygen and nutrients to the rapidly growing tumor47. The interplay between VEGFR-2 and Tie2 signaling is crucial for the proper formation of blood vessels in tumor vasculature.
Although Tie2 and VEGFR2 are primarily expressed on endothelial cells, aggressive TNBC cells can aberrantly express these angiogenic receptors48. Indeed, mechanisms that support feeding and oxygen delivery to tumor cells, such as VM, contribute to tumor growth. Ectopic expression facilitates tumor-endothelial mimicry, a phenomenon in which cancer cells acquire endothelial-like traits and form vascular structures independently of endothelial cells (VEGF-independent). Vasculogenic mimicry is linked to increased invasiveness, metastasis, and resistance to anti-angiogenic treatments. Investigating Tie2 and VEGFR2 expression in this context also provides valuable insights into autocrine and paracrine VEGF signaling within the tumor microenvironment (TME), offering promising therapeutic targets for TNBC22,49.
In this study, we evaluated the expression of Tie2 and VEGFR2 using real-time PCR. Our results revealed that RT alone did not significantly alter their expression levels in both MDA-MB-231 and MDA-MB-468 cells, whereas Ag-NPs reduced the levels of these angiogenic factors. Notably, combination therapy with Ag-NPs and RT further intensified this downregulation, possibly by modulating tumor cell plasticity or angiogenic mimicry.
MMPs are a family of proteolytic enzymes responsible for extracellular matrix (ECM) degradation, thereby facilitating tumor invasion, metastasis, and overall malignant progression50. Among them, MMP-9 and MMP-2 are the most prominent members of MMPs that play integral roles in ECM remodeling, which enables cancer cell migration and metastatic spread51. According to the literature, RT can exert both pro-angiogenic and pro-metastatic effects, depending on the TME, radiation dose, and cancer type. While sub-lethal doses of RT can enhance angiogenesis and metastatic potential in some cancers52,53, higher doses are more likely to damage tumor vasculature and suppress metastasis54. Although several preclinical models have demonstrated radiation-induced enhancement of metastatic disease, clinical evidence in humans remains inconsistent, likely due to tumor heterogeneity and context-dependent biological responses55. For instance, in a mouse xenograft model, rhabdomyosarcoma cell lines derived from two different patients exhibited opposite effects of fractionated irradiation on metastatic spread, highlighting the complexity and variability of tumor responses to RT56. A deeper understanding of the mechanisms underlying the observed effects in our study is essential to identify those most relevant to human RT scenarios and to elucidate the precise pathways that may contribute to radiation-induced metastasis.
In this study, western blotting revealed that both Ag-NP and RT therapy significantly reduced the protein levels of MMP9, pro-MMP2, and cleaved-MMP2 (activated-MMP2) in MDA-MB-231 cell line. Notably, a combination therapy resulted in a more pronounced reduction in MMP9 expression and a lower cleaved-MMP2/pro-MMP2 ratio than either monotherapy. This discrepancy with the existing literature may be due to various factors, including radiation dose, variations in the TME, and potential involvement of specific signaling pathways. While we acknowledge that protein expression does not fully confirm enzymatic activity, the functional effects observed in tube formation assays support impairment of invasion- and angiogenesis-related processes.
We also employed a scratch assay to evaluate the effects of monotherapies and combination therapy on cancer cell migration. The results revealed a notable reduction in cell migration and slower wound-healing rates in treated cells, suggesting that Ag-NPs effectively inhibit MDA-MB-231 cell migration. These findings underscore the potential of Ag-NPs as a promising therapeutic agent for mitigating metastasis in TNBC cell lines.
To further assess the anti-angiogenic potential of Ag-NPs, a tube formation assay was conducted using endothelial cells co-cultured with the MDA-MB-231 cell line. Due to the inherent limitations of the scratch assay, where wound closure can result from both cell migration and proliferation, this complementary assay was employed to reinforce our findings. Quantitative analysis demonstrated that both Ag-NPs and RT alone significantly reduced the number of junctions compared with the control group. Notably, the combination of Ag-NPs with RT led to a much stronger inhibitory effect on tube formation, indicating a potent synergistic suppression of angiogenesis-related processes. Given the close link between angiogenesis and metastatic progression, the observed inhibition of tube formation, together with the downregulation of VEGFR2, Tie2, and MMP-2/9, further supports that Ag-NPs combined with RT exert both anti-angiogenic and anti-metastatic effects.
The clinical significance of this synergistic interaction remains to be validated in vivo, where factors such as pharmacodynamics, TME, and radiation fractionation may influence therapeutic outcomes.
Nevertheless, the rigorous synergy quantification provides a strong preclinical basis for further translational investigation.

Conclusion
This study demonstrates that Ag-NPs act as a potent radiosensitizer in TNBC, enhancing RT-induced cytotoxicity through increased ROS generation and apoptosis. The combination of Ag-NPs with RT effectively disrupted key angiogenic and migratory pathways by downregulating MMP-2, MMP-9, VEGFR-2, and Tie2, resulting in impaired ECM remodeling, significant reduction in cell migration, and a decreased number of junctions in tube formation assays, indicating inhibition of both angiogenic and vasculogenic activity.
These findings highlight the dual anti-angiogenic and anti-migratory effects of Ag-NPs, suggesting their potential to improve therapeutic efficacy in TNBC. Nevertheless, as these results are derived from in vitro experiments, further in vivo studies are required to validate these effects and elucidate the underlying molecular mechanisms supporting clinical translation of Ag-NPs as a radiosensitizer. Additionally, the precise source of the increased ROS remains to be determined. Future studies employing targeted inhibition or knockdown of Tie2 and VEGFR2 are needed to clarify their mechanistic involvement in the observed anti-angiogenic, anti-vasculogenic mimicry, and radiosensitizing effects.

Supplementary Information
Below is the link to the electronic supplementary material.