Dual-transferred atmospheric-pressure plasma jet modulates matrix metalloproteinase expression in breast cancer stem cells

TL;DR

A new low-power plasma jet system effectively reduces breast cancer stem cell activity by suppressing genes linked to tumor spread.

Contribution

A dual-transferred atmospheric-pressure plasma jet (DTAPPJ) system is introduced for targeted suppression of breast cancer stem cells via matrix metalloproteinase modulation.

Findings

Helium-based DTAPPJ more effectively suppresses multiple MMP genes compared to argon.

DTAPPJ reduces breast cancer stem cell viability and metabolic activity in a time- and gas-dependent manner.

Gas-specific reactive oxygen and nitrogen species profiles drive the anticancer effects of DTAPPJ.

Abstract

Breast cancer stem cells (BCSCs) are a highly aggressive subpopulation, driving tumor initiation, metastasis, and therapeutic resistance, largely through matrix metalloproteinases (MMPs)–mediated extracellular matrix remodeling. Here, we introduce a dual-transferred atmospheric-pressure plasma jet (DTAPPJ) platform designed to enhance plasma stability, reactive species delivery, and spatial controllability, enabling targeted modulation of BCSCs. The DTAPPJ system was evaluated using argon and helium as working gases, with direct plasma exposure for 120, 180, and 240 s. Voltage–power measurements confirmed stable plasma propagation and low energy consumption (<1 W). At the same time, optical emission spectroscopy revealed a sequential amplification of reactive oxygen and nitrogen species (RONS) across primary, secondary, and tertiary jets. Biologically, DTAPPJ exposure induced robust, time-dependent suppression of multiple MMP genes, including MMP-1, -2, -3, -7, -9, -10, −11, −13, and −14, with helium-driven plasma consistently outperforming argon. This repression correlated with significant reductions in BCSC viability and metabolic activity in the 3D culture system, highlighting the system's ability to overcome intrinsic antioxidant defenses. Mechanistically, DTAPPJ likely exerts its effects through the oxidative modulation of redox-sensitive transcription factors that regulate MMP expression, thereby collectively diminishing the invasive and metastatic potential. The dual-transfer architecture, incorporating floating copper electrodes, ensures safe operation, enhanced reactive species generation, and minimal thermal impact. Overall, our findings demonstrate that DTAPPJ represents a low-power, multi-targeted plasma modality capable of simultaneously suppressing MMP-mediated invasion and impairing BCSC viability, providing a promising strategy for translational anticancer applications and potential minimally invasive therapies.

Image 1

•DTAPPJ delivers stable, low-power plasma with rich RONS profiles from Ar and He gases.•Helium-based DTAPPJ consistently suppresses MMP genes more strongly than argon.•Prolonged exposure to DTAPPJ (240 s) maximizes the inhibition of MMPs transcript levels.•DTAPPJ markedly reduces BCSC viability in a time-dependent, gas-dependent manner.•Optical emission shows gas-specific RONS diversity driving DTAPPJ anticancer effects.

DTAPPJ delivers stable, low-power plasma with rich RONS profiles from Ar and He gases.

Helium-based DTAPPJ consistently suppresses MMP genes more strongly than argon.

Prolonged exposure to DTAPPJ (240 s) maximizes the inhibition of MMPs transcript levels.

DTAPPJ markedly reduces BCSC viability in a time-dependent, gas-dependent manner.

Optical emission shows gas-specific RONS diversity driving DTAPPJ anticancer effects.

Figures

DTAPPJ schematic diagram.Fig. 1

Schematic representation of RONSs identified by OES for the DTAPPJ system: (a) argon-driven DTAPPJ in the first APPJ; (b) helium-driven DTAPPJ in the first APPJ; (c) argon-driven DTAPPJ during the first transfer stage (second APPJ); (d) helium-driven DTAPPJ during the first transfer stage (second APPJ); (e) argon-driven DTAPPJ during the second transfer stage (third APPJ); and (f) helium-driven DTAPPJ during the second transfer stage (third APPJ).Fig. 3

Analysis of MMP-1 expression following DTAPPJ exposure using (a) argon, (b) helium, and (c) a direct comparison between the two gas types (*P = 0.0006). Assessment of MMP-2 expression under DTAPPJ treatment using (d) argon, (e) helium, and (f) a comparative view of both gases (P = 0.0011). Assessment of MMP-3 expression after DTAPPJ application using (g) argon, (h) helium, and (i) a side-by-side comparison of argon-versus helium-based treatment (*P = 0.0005).Fig. 4Fig. 5Analysis of MMP-7 expression following DTAPPJ exposure using (a) argon, (b) helium, and (c) a direct comparison between the t

Assessment of BCSC viability following DTAPPJ treatment: (a) argon, (b) helium, and (c) direct comparison between argon- and helium-based plasma.Fig. 7

Effects of DTAPPJ on the parameters evaluated in this investigation.Fig. 8

Full text

Introduction

1

Breast cancer remains one of the most prevalent and lethal malignancies among women worldwide [1]. Despite advances in early detection, targeted therapy, and personalized medicine, it continues to cause significant morbidity and mortality [2]. Its clinical complexity arises from profound heterogeneity across molecular subtypes, including luminal A, luminal B, HER2-enriched, and triple-negative tumors, each driven by distinct oncogenic pathways and microenvironmental dependencies [3,4]. Metastasis is the leading cause of breast cancer–related mortality [5]. It involves a multistep process encompassing local invasion, extracellular matrix (ECM) degradation, intravasation into the vasculature, survival in circulation, extravasation, and colonization of distant organs [[6], [7], [8]]. Central to this process is ECM remodeling, predominantly orchestrated by matrix metalloproteinases (MMPs)—zinc-dependent endopeptidases that degrade structural and signaling components of the ECM [9]. Under physiological conditions, MMPs are tightly regulated and contribute to tissue homeostasis, including wound healing, morphogenesis, and mammary gland involution [10]. In cancer, this regulatory control is lost. Overexpression of specific MMPs drives tumor invasion, angiogenesis, and metastasis [11,12]. In breast cancer, a defined subset of MMPs—including MMP-1, -2, -3, -7, -9, -11, −13, −14, −19, −21, −23, −26, and 28—has been consistently associated with aggressive phenotypes, poor prognosis, and enhanced metastatic potential [[13], [14], [15]]. These enzymes degrade key ECM components such as collagen, laminin, fibronectin, and proteoglycans [16]. Beyond proteolysis, they modulate growth factor availability, cytokine signaling, and cell–cell adhesion, promoting epithelial-to-mesenchymal transition (EMT), stemness, and resistance to apoptosis [17,18]. The ECM is a dynamic entity that provides both mechanical support and biochemical cues [19]. Its hydrated, viscoelastic phase—termed the “fundamental sol”—comprises collagen fibers, glycoproteins, and proteoglycans, forming a scaffold that regulates adhesion, migration, and signal transduction [20,21]. In breast cancer, excessive MMP activity leads to pathological ECM stiffening, altered mechanotransduction, and enhanced integrin signaling, which together facilitate invasive behavior [22].

MMP-1 initiates interstitial collagen cleavage and establishes primary ECM remodeling [23]. MMP-2 plays a central role in breast cancer progression [24]. It degrades basement-membrane collagen and enhances invasive capacity [25]. It also contributes to breast cancer stem cell (BCSC) survival by engaging PI3K/AKT and STAT3 signaling. MMP-3 enhances this activity by activating downstream proteases [26]. MMP-7 disrupts epithelial adhesion through E-cadherin cleavage and facilitates early EMT [27]. MMP-9, produced by tumor-associated macrophages and BCSCs, degrades type IV collagen and releases VEGF, thereby supporting angiogenic signaling [28]. MMP-11, secreted in its active form by CAFs, modulates stromal architecture and protects malignant cells from apoptosis [29]. MMP-13 intensifies collagen turnover and promotes structural permissiveness for invasion [30]. MMP-14 (MT1-MMP) functions as a membrane-anchored protease that drives pericellular matrix degradation and activates pro-MMP-2 [31]. MMP-19 and MMP-21 regulate angiogenic and immune-modulatory cues within the tumor microenvironment [32]. MMP-23, MMP-26, and MMP-28 support epithelial proliferation and invasion through combined enzymatic and signaling-dependent mechanisms [33]. Collectively, these thirteen MMPs form a coordinated proteolytic network [34]. They destabilize ECM integrity, modulate its biomechanical properties, and establish a feed-forward axis that promotes inflammation, stemness, and directional invasion [35]. Subsequently, BCSCs represent a highly aggressive subpopulation responsible for metastasis, recurrence, and therapeutic resistance [36]. They occupy ECM niches enriched in MMP activity, which preserve stemness features and facilitate directed migration [37]. Elevated expression of MMP-2, MMP-9, and MMP-14 in BCSCs accelerates ECM degradation and augments survival signaling [38]. The reciprocal interactions between BCSCs, MMPs, and the ECM generate a self-sustaining microenvironmental circuit that drives metastatic dissemination [39]. Thus, developing new strategies that suppress MMPs to eliminate BCSCs appears essential, and cold atmospheric plasma (CAP) represents a promising, non-invasive, and efficient option [40].

CAP has recently emerged as a promising non-thermal modality capable of targeting this axis [41]. CAP is a partially ionized gas generated at atmospheric pressure and near-room temperature [42]. It contains reactive oxygen and nitrogen species (RONS), electrons, ions, UV photons, and transient electric fields [43]. When applied to biological systems, CAP delivers reactive species, including hydrogen peroxide (H_2_O_2_), hydroxyl radicals (•OH), and nitric oxide (NO), which penetrate cells and tissues to induce redox signaling [44]. At controlled doses, these species modulate signaling cascades; at higher doses, they induce oxidative stress, DNA damage, and apoptosis [45]. Cancer cells, including BCSCs, are particularly susceptible due to elevated basal oxidative stress and weaker antioxidant defenses [46]. CAP has been shown to inhibit proliferation, migration, and invasion in breast cancer models [47]. Mechanistically, it downregulates pro-metastatic MMPs, particularly MMP-2, -9, and -14, by inhibiting redox-sensitive transcription factors such as NF-κB and STAT3 [48]. This reduces ECM degradation, impairs invadopodia formation, and diminishes angiogenic signaling [49]. CAP also modifies ECM mechanics by inducing oxidative cross-linking of collagen and proteoglycans, thereby decreasing stiffness and porosity, which in turn constrains tumor cell migration [50,51]. These effects help restore the balance between MMPs and their inhibitors (TIMPs), stabilizing ECM integrity [52,53]. The transient electric fields in CAP further influence cell membrane potential and mechanotransduction, disrupting integrin clustering and focal adhesion dynamics [54,55]. Beyond these local effects, CAP can promote anti-tumor immunity by inducing immunogenic cell death and the release of danger-associated molecular patterns (DAMPs), leading to activation of cytotoxic T lymphocytes and further suppression of MMP expression and pro-metastatic inflammatory signaling [[56], [57], [58]].

CAPs employed in biomedical and clinical applications encompass a broad spectrum of source configurations, including dielectric barrier discharges (DBDs), atmospheric-pressure plasma jets (APPJs), and transferred plasma systems, each differing in plasma chemistry, power coupling, and interaction mechanisms with biological tissues [59]. Among these platforms, transferred atmospheric-pressure plasma jets (T-APPJs) are of particular interest because they enable a clear spatial separation between the plasma generation zone and the treatment site, which is a critical requirement for safe medical use [60]. This configuration allows plasma-derived RONSs, energetic electrons, and transient electric fields to be delivered remotely to biological targets without exposing tissues to high voltages or excessive heat [61,62]. Compared with conventional APPJs, T-APPJs exhibit superior penetration and diffusion of reactive species into narrow, enclosed, or porous biological environments—such as tissue crevices, wound beds, and tumor-associated extracellular matrices—while maintaining near-room-temperature operation and minimizing the risk of electrical shock or thermal damage [63]. A central design element in many T-APPJ systems is the incorporation of a copper wire inside the transfer tube acting as a floating electrode, which plays a crucial role in shaping the local electric field and sustaining plasma propagation along the tube [64]. The floating copper wire enhances plasma stability, increases jet length, and promotes higher RONS density by facilitating charge accumulation and redistribution without direct electrical coupling to the target [65]. Collectively, these characteristics provide greater operational flexibility, improved safety, and enhanced biological efficacy, making T-APPJs particularly well-suited for precision plasma medicine applications, including antimicrobial treatment and cancer therapy [66,67].

This study investigates the effects of a novel transmissional CAP platform, termed the dual-transferred atmospheric-pressure plasma jet (DTAPPJ), on MMPs associated with BCSCs, including MMP-1, -2, -3, -7, -9, -10, −11, −13, and −14. These enzymes play pivotal roles in extracellular matrix remodeling and tumor invasiveness. Compared to conventional APPJs, DTAPPJ offers several advantages, including physical separation between plasma generation and treatment regions, enhanced penetration and diffusion into confined microenvironments, high operational flexibility, and improved treatment safety. These advantages are achieved by reducing the applied voltage while simultaneously enhancing the production and reactivity of plasma-derived RONSs, thereby minimizing the risk of electrical shock and thermal damage to biological tissues. By elucidating the molecular mechanisms through which DTAPPJ modulates MMP expression and activity, this study aims to provide mechanistic insights that may support the development of innovative therapeutic strategies for selectively targeting BCSCs and improving breast cancer treatment outcomes.

Methods

2

Materials

2.1

All experiments involving BCSCs were conducted using the MCF7 human breast carcinoma line, obtained from the NCBI Cell Repository at the Pasteur Institute of Iran. Three-dimensional spheroid cultures were established in the Matrigel matrix (Corning, AZ, USA). Cells were seeded and maintained in 96-well and 48-well culture plates (Nunc, Denmark; Shanghai Biotechnology, China). The culture environment comprised DMEM/F12 medium supplemented with streptomycin, B27 (Gibco, USA), recombinant human EGF, leukemia inhibitory factor, and insulin (Sigma, USA), basic FGF (Upstate Products, USA), and vincristine (Shanghai Hualin Pharmaceutical Co., China). Cell viability was quantified via absorbance readings obtained from an ELISA microplate reader (Model XYZ, Roche Applied Sciences, Indianapolis, USA). RNA extraction and cDNA amplification for gene expression profiling were performed using the RNA PCR Kit version 3.0 (TaKaRa, Japan).

For the generation of the translational CAP system, a variable-frequency AC power source (Model LF-HV, Basafn, Tehran, Iran) was employed. Plasma diagnostics were performed using an Ocean Optics USB4000 spectrometer (Orlando, FL, USA), a Tektronix TDS2024D oscilloscope (Beaverton, OR, USA), and a high-voltage probe from Siglent Technologies (Ohio, USA). Argon and helium, serving as the carrier gases, were obtained from Sepehr Gas Kavian (Tehran, Iran). The plasma jet apparatus consisted of quartz discharge tubes (Iran Quartz, Tehran, Iran), PTFE insulation (Sepahan Polymer, Isfahan, Iran), copper electrodes (Mehr Asl, East Azerbaijan, Iran), and flexible polymer tubing (Persian Sanat, Tehran, Iran).

Methods

2.2

This research explored how a DTAPPJ influences the expression of MMP genes in BCSCs cultured in a three-dimensional system. The DTAPPJ was generated by applying a high alternating current voltage to a flow of argon and helium gases, producing plasma in two successive, continuous discharge phases. The resulting plasma was then applied to BCSCs for exposure durations of 120, 180, and 240 s. Following treatment, quantitative real-time PCR was employed to determine the transcriptional changes in MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, MMP-11, MMP-13, and MMP-14. Furthermore, the metabolic activity and survival of BCSCs post-exposure were examined through the MTT colorimetric assay.

Cell culture in Matrigel

2.2.1

BCSCs isolated from the MCF7 cell line were cultured using RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics—penicillin (100 U/mL) and streptomycin (100 μg/mL). Cultures were maintained at 37 °C in a humidified incubator containing 5% CO_2_ and 95% air. For 3D growth, each well of a pre-chilled 48-well plate received 200 μL of Matrigel, which was allowed to polymerize at 37 °C for 30 min. After solidification, the surface was leveled with a pipette tip. Subsequently, BCSCs were plated at a density of 2 × 10^4^ cells per well and incubated for 14–18 h in 500 μL of Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS, penicillin G (100 U/mL), and streptomycin (100 μg/mL). After this period, 250 μL of the medium was withdrawn and replaced with an equal volume of serum-free cancer stem cell medium formulated from DMEM/F12 containing leukemia inhibitory factor (10 ng/mL), basic fibroblast growth factor (20 ng/mL), epidermal growth factor (20 ng/mL), B27 supplement, insulin (4 U/L), and vincristine (5 ng/mL). This medium exchange step was then performed once more to maintain optimal culture conditions.

Dual-transferred atmospheric-pressure plasma jet

2.2.2

The generation of the DTAPPJ, depicted schematically in Fig. 1, involves a sequential three-stage configuration. To begin with, an APPJ is produced using helium and argon as carrier gases under a high-voltage alternating current. The plasma discharge occurs between two copper electrodes: the first is a cylindrical tube (5 cm long, 6 mm inner diameter, 8 mm outer diameter), and the second is an O-shaped ring electrode (3 mm thick, 8 mm inner diameter, 9 mm outer diameter). These electrodes are separated by a 10 cm quartz tube (8 mm ID, 10 mm OD).Fig. 1DTAPPJ schematic diagram.Fig. 1

Attached to the downstream end of this quartz segment is a flexible plastic conduit 6 m in length with internal and external diameters of 4 mm and 6 mm, respectively. Inside this conduit lies an unconnected (floating) copper filament, also 6 m long and 0.1 mm in diameter, which begins a few millimeters downstream from the initial APPJ zone and terminates just before the outlet of the tube. Although electrically isolated, this wire enhances ionization within the gas flow, enabling the plasma plume to extend through the entire length of the tube. In the second phase, the electric field generated at the wire's free end initiates a secondary discharge at the tube outlet—constituting the first plasma transfer. This section is then joined to a second plastic conduit by a short quartz linker (5 cm long, 4 mm ID, 6 mm OD). The second conduit, structurally identical to the first, contains another thin copper wire spanning its full length to sustain plasma propagation. Finally, in the third stage, the secondary jet is transmitted through this second conduit in the same manner, where the induced electric field again drives plasma formation at the distal end, completing the process and yielding the dual-transferred cold atmospheric plasma (second transfer).

The DTAPPJ system was powered by a variable-frequency, half-bridge AC high-voltage supply that generated an oscillating potential of approximately 20 kV peak-to-peak with a maximum current of about 500 mA. Electrical characterization of the plasma discharge was performed through simultaneous measurements of voltage, current, transferred charge, and discharge power using a Tektronix TDS2024D oscilloscope. The applied high voltage was monitored at the generator output via a high-voltage probe connected through a 1:1000 voltage divider. To assess voltage transmission along the system, an additional high-voltage probe was employed to measure the voltage at the distal end of the floating copper wires. Two high-voltage probes (100 MΩ, 1000X, 5 pF, 40 MHz), together with a conventional 100X probe, were used to capture and analyze the voltage and current waveforms of the plasma jet.

The discharge current was indirectly determined by measuring the voltage drop across a grounded series shunt resistor (10 kΩ) integrated into the circuit. For charge transfer analysis, an external capacitor (27 nF) was connected in series with the plasma device, and the voltage across this capacitor was recorded to calculate the transferred charge. The plasma power consumption was evaluated using the Lissajous (Q–V) method, in which the energy dissipated per voltage cycle was obtained from the area enclosed by the Q–V loop formed by plotting the transferred charge against the applied voltage. The average discharge power was then calculated accordingly.

Optical emission spectroscopy (OES) was conducted to characterize DTAPPJ reactive species using an Ocean Optics USB4000 spectrometer operating over the wavelength range of 300–1100 nm with an integration time of 1000 ms. Spectral measurements were performed for each of the three plasma jets at a fixed distance of 3 cm from the jet outlet. Initially, optical emission spectra were recorded for the first APPJ operating independently. Subsequently, spectra corresponding to the second APPJ during the first transition state were obtained, followed by spectral acquisition of the APPJ during the second transition state.

In vitro assay of cell viability

2.2.3

The metabolic viability of BCSCs subjected to DTAPPJ exposure was assessed through the MTT colorimetric assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). To ensure reproducibility, each experimental condition was analyzed in triplicate. Briefly, 5 × 10^3^ cells were seeded onto sterile scaffolds placed in individual wells of 96-well culture plates. After incubation for 24, 48, and 72 h, a 5 mg/mL MTT stock solution was added to each well to evaluate mitochondrial enzymatic activity as an indicator of cellular metabolism. The cultures were then maintained for an additional 24 h at 37 °C in a humidified incubator containing 5% CO_2_. Control wells containing untreated BCSCs in a standard culture medium were processed under identical conditions. Following incubation, the medium was discarded, and 100 μL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals formed by metabolically active cells. Absorbance was measured at 570 nm using a microplate spectrophotometer, and cell viability was calculated using the following conventional formula to compare plasma-treated samples with untreated controls:

$$[eqn]$$

Gene expression analysis by RT-PCR

2.2.4

Real-time PCR was employed to examine the transcriptional activity of ATP-binding cassette (ABC) transporter genes associated with vincristine efflux mechanisms. Total RNA was extracted from MCF7-derived cell fractions—comprising both adherent monolayer cultures and spheroid-forming stem-like tumor cells—using the Tripure isolation reagent according to the supplier's protocol. Complementary DNA (cDNA) was synthesized and amplified with the TaKaRa RNA PCR Kit version 3.0. Each target gene was amplified through 29 thermal cycles, while glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal reference for expression normalization. The amplified products were separated on a 1.8% agarose gel containing 0.5 mg/mL ethidium bromide and visualized using ultraviolet transillumination.

Statistical analysis

2.2.5

A p < 0.05 was considered statistically significant for all comparisons between treated and control groups. Statistical processing of the data was conducted using GraphPad Prism version 9.0, with all measurements expressed as mean ± standard deviation (SD). Depending on the experimental design, either one-way or two-way analysis of variance (ANOVA) was utilized to assess group differences, followed by Tukey's post-hoc test to determine pairwise significance among multiple conditions.

Results and discussion

3

Breast cancer progression and metastasis are critically driven by MMPs, particularly MMP-2 and MMP-9, which degrade ECM components, facilitate tumor cell invasion, promote angiogenesis, and enable metastatic dissemination [68]. In BCSCs, MMP activity is tightly linked to stemness maintenance, EMT, and enhanced migratory capacity, thereby amplifying their tumor-initiating and metastatic potential [69]. BCSCs represent a clinically significant subpopulation characterized by elevated antioxidant capacity, self-renewal potential, and a disproportionate contribution to metastasis and tumor recurrence [70]. These cells maintain suppressed basal reactive oxygen species (ROS) levels, enabling survival under oxidative and genotoxic stresses that typically eliminate non-stem cancer cells [71]. Moreover, BCSCs actively remodel the ECM and invade restrictive microenvironments, which enhances their metastatic competence and resistance to conventional therapies [72]. Given the central role of MMPs in breast cancer progression, multiple therapeutic strategies have been developed to target their expression or activity [73]. Although preclinical studies have demonstrated reduced invasion and metastasis following MMP suppression, clinical translation has been limited by issues including lack of specificity, dose-limiting toxicity, and compensatory activation of alternative proteolytic pathways [74,75]. In this context, CAP has emerged as a promising therapeutic modality for targeting BCSCs by generating RONSs that modulate intracellular signaling, induce oxidative stress, and suppress stemness- and invasion-associated programs, including MMPs expression and activity [[76], [77], [78], [79]]. However, conventional CAP systems often suffer from limited spatial reach, high energy requirements, or insufficient penetration into complex 3D microenvironments, restricting their potential for translational applications [80].

To overcome the limitations associated with conventional CAP systems, we employed a newly developed DTAPPJ platform designed to enhance plasma stability, spatial controllability, and reactive species delivery efficiency (Fig. 1). Unlike traditional CAP jets, where electron density and reactive species decay monotonically with distance [81], DTAPPJ maintains plasma effectiveness by reinitiating discharge at the transfer stages, thereby compensating for losses typically associated with long paths. In this study, the DTAPPJ system was utilized to selectively target BCSCs and to evaluate both cellular viability and the suppression of BCSC-associated MMPs. Direct plasma exposure was applied for 120, 180, and 240 s. Plasma generation was initiated through an electrical discharge between the inner and outer electrodes, followed by sequential plasma transfer through two independent channels, resulting in the emission of APPJs from dual transmission outlets. Stable plasma propagation was achieved using argon and helium as working gases, sustained by high-voltage alternating current delivered via copper wires embedded within the transfer tubes. These wires functioned as floating electrodes, guiding plasma propagation, shaping the electric field distribution, improving discharge stability, and enhancing reactive species production. The primary APPJ served as the initial source of ionized gas, while the transferred plasma sequentially ignited secondary and tertiary jets, with the final transferred jet directly irradiating the cultured BCSCs. The DTAPPJ system was operated at applied voltages of 9.72 kV for argon and 8.85 kV for helium, with corresponding discharge currents of 4.4 mA and 4.26 mA, respectively. In both cases, the gas flow rate was maintained at 3 L/min, and the operating frequencies were 40.33 kHz for argon and 37.54 kHz for helium (Fig. 2a and b). As shown in Fig. 2a and b, the voltage and current waveforms exhibited a pronounced attenuation along the plasma transfer path, indicating stable discharge conditions and effective energy transmission through the system. At the end of the primary copper wire, the voltage and current decreased to 1.247 kV and 3.39 mA for argon and to 1.167 kV and 3.28 mA for helium. Further attenuation was observed at the termination of the secondary copper wire, where the voltage and current were reduced to 0.291 kV and 2.72 mA for argon and to 0.263 kV and 2.98 mA for helium, respectively. This progressive voltage and current drop is mainly attributed to energy dissipation during gas ionization processes, which simultaneously contributes to reduced overall power consumption [82]. In addition, the plasma column formed between the two copper wires introduces an effective electrical resistance, functioning as a resistive load during discharge initiation and propagation [83]. This resistive behavior further explains the observed voltage and current attenuation. Such performance aligns with prior studies demonstrating that wire-guided plasma transfer can preserve discharge stability over extended lengths if appropriately impedance-matched and grounded [84].Fig. 2(a) Voltage and current waveforms of the argon DTAPPJ; (b) voltage and current waveforms of the helium DTAPPJ; (c) Lissajous (Q–V) plot corresponding to one voltage period during the second transfer in the argon-gas DTAPPJ; and (d) Lissajous (Q–V) plot corresponding to one voltage period during the second transfer in the helium-gas DTAPPJ.Fig. 2

In addition to these voltage–power characteristics, the safety of the DTAPPJ system was further evaluated by calculating the consumed power using Q–V plots (i.e., Lissajous figures). Specifically, the output plasma jet power (P) was determined using the relation

$$[eqn]$$

where V represents the plasma jet voltage, q denotes the charge measured across the external capacitor, and f is the signal frequency. Based on this calculation, the output plasma jet power was determined to be 0.792 W for argon and 0.786 W for helium (Fig. 2c and d). Both values are below 1 W, providing further evidence of the system's low energy demand and safe operational characteristics. The term “safety” specifically refers to the minimization of electrical shock and thermal hazards for both the operator and the treated site (human or animal). Owing to the extremely low delivered power, the risk of unintended electrical discharge, excessive current flow, or Joule heating is reduced to the lowest practical level. Consequently, the plasma treatment remains electrically benign and thermally gentle, even during prolonged exposure, ensuring safe system operation under biomedical conditions. Taken together, the observed voltage drops, low output power below 1 W, and enhanced plasma stability represent key advantages of the DTAPPJ system compared with conventional APPJs, ensuring safer operation and reduced thermal and electrical risks.

RONS generated by the DTAPPJ—central mediators of its anticancer effects—were analyzed using optical emission spectroscopy (OES), with measurements conducted 3 cm from each APPJ (Fig. 3a–f). Across all operating conditions, the dominant emission features observed in the 300–400 nm spectral region originated primarily from the second positive system (SPS) of molecular nitrogen (N_2_: C^3^Πᵤ → B^3^Πg). This behavior is consistent with atmospheric-pressure plasma jets, in which molecular nitrogen from ambient air constitutes the most abundant radiative species. In contrast, emission from atomic nitrogen and ionic species is expected to be weak due to their low steady-state concentrations and rapid recombination kinetics at atmospheric pressure. A progressive increase in nitrogen-related emission features was observed along successive plasma transfer stages. This trend is most plausibly attributed to minor air entrainment into the plasma channel, which becomes more pronounced with increasing jet length. Such air leakage is inherent to open or semi-open atmospheric plasma configurations and does not negatively affect plasma stability or performance. Instead, it contributes to enhanced formation of reactive nitrogen species (RNS), which are biologically relevant for plasma–cell interactions. The primary argon-fed APPJ produced emission lines of Ar I, O I, N I, and Cu I (Fig. 3a), whereas the primary helium-fed APPJ exhibited He I, O I–II, N_2,_ N I, and Cu I (Fig. 3b). The secondary APPJ with argon displayed Ar I–II, O I–II, N_2_, N I–II, Cu I–II, and molecular ions including O_2_^+^, N_2_^+^, and NO (Fig. 3c), while the helium-fed secondary APPJ showed He I, O I–II, N_2,_ N I–II, Cu I–II, and the same molecular ions (Fig. 3d). The tertiary APPJ with argon produced Ar I–III, O I–III, N_2,_ N I–III, Cu I–II, and molecular ions O_2_^+^, N_2_^+^, NO, and N^+^ (Fig. 3e), whereas the tertiary helium-fed APPJ exhibited He I, O I–IV, N_2,_ N I–IV, Cu I–II, and molecular ions O_2_^+^, N_2_^+^, NO, and N^+^ (Fig. 3f). These reactive species, particularly O I–IV, N_2,_ N I–III, and molecular ions such as O_2_^+^, N_2_^+^, NO, and N^+^, are the main RONS responsible for BCSC cytotoxicity. They collectively drive oxidative and nitrosative signaling, induce DNA damage, disrupt mitochondrial function, and activate apoptotic pathways, ultimately leading to the death of effective BCSCs [85,86]. These observations indicate a significant increase in both the number and intensity of reactive species at each plasma transfer stage, with the tertiary APPJ generating a notably richer RONS profile than the preceding jets. This enhancement is likely attributable to the sequential ionization of argon and helium particles across the three stages: initial ionization in the primary APPJ, subsequent ionization in the secondary jet, and final ionization in the tertiary jet. Such cumulative ionization markedly amplifies reactive species production, representing a key advantage of the DTAPPJ over conventional APPJs and enabling effective oxidative signaling over extended distances, even under low applied power conditions. Notably, the greater reactive output observed in helium-driven configurations arises from discharge physics rather than gas chemistry; helium sustains a higher electron energy distribution and electron density under identical operating conditions, facilitating more efficient energy transfer to ambient air molecules, enhanced Penning ionization, and improved propagation stability, which collectively promote the formation and transport of long-lived RONS [87,88]. Upon interaction with the liquid phase of the cell culture medium, DTAPPJ-generated species such as O, N, O_2_^+^, N_2_^+^, and NO initiate secondary aqueous chemistry. Oxygen-derived species form hydroxyl radicals (•OH) and superoxide (O_2_•^-^), which subsequently recombine or undergo dismutation to yield hydrogen peroxide (H_2_O_2_) [89]. Concurrently, nitrogen species, including NO and excited N atoms, dissolve into the medium and undergo oxidation in the presence of dissolved oxygen and ROS to form NO_2_, which is further converted into nitrite (NO_2_^−^) [90]. These long-lived species readily diffuse into aqueous microenvironments, sustaining oxidative pressure sufficient to perturb intracellular redox networks [91,92]. Thus, the preservation of strong molecular nitrogen emission over extended transfer distances demonstrates that the DTAPPJ maintains chemically active plasma conditions despite operating at ultra-low power, highlighting its suitability for safe and effective biomedical applications.Fig. 3. Schematic representation of RONSs identified by OES for the DTAPPJ system: (a) argon-driven DTAPPJ in the first APPJ; (b) helium-driven DTAPPJ in the first APPJ; (c) argon-driven DTAPPJ during the first transfer stage (second APPJ); (d) helium-driven DTAPPJ during the first transfer stage (second APPJ); (e) argon-driven DTAPPJ during the second transfer stage (third APPJ); and (f) helium-driven DTAPPJ during the second transfer stage (third APPJ).Fig. 3

In addition to oxidative stress, electrostatic effects may also contribute to DTAPPJ-mediated cytotoxicity [93]. Local electric fields generated by charged species at the plasma–liquid interface can perturb the cell membrane, alter ion transport, and enhance intracellular stress [94,95]. These electrostatic interactions can act synergistically with RONS to amplify DNA damage, mitochondrial disruption, and apoptosis, as reported in previous studies [[96], [97], [98]]. While direct measurement of electrostatic stress was beyond the scope of this study, these complementary effects likely enhance the overall cytotoxic efficiency of DTAPPJ.

Biologically, DTAPPJ exposure induced robust and statistically significant suppression of multiple MMP genes in BCSCs (Fig. 4, Fig. 5, Fig. 6). MMPs orchestrate ECM remodeling, basement membrane degradation, and metastatic invasion in breast cancer [99]. Collagenases such as MMP-1 and MMP-13, which drive stromal remodeling [100], were strongly reduced (***P = 0.0006, Fig. 4a–c; ***P = 0.0002, Fig. 6d–f), indicating that DTAPPJ impairs key collagenolytic pathways. Gelatinases MMP-2 and MMP-9, central to basement membrane dissolution and invasion competence [101], were similarly downregulated (**P = 0.0011, Fig. 4d–f; ***P = 0.0005, Fig. 5d–f). Membrane-bound MMP-14 (MT1-MMP), essential for pro-MMP-2 activation and local ECM degradation [102], was also markedly inhibited (**P = 0.0016, Fig. 6g–i), suggesting that DTAPPJ disrupts both catalytic activity and upstream protease activation cascades. Additional MMPs, including MMP-3 (***P = 0.0005), MMP-7 (***P = 0.0003), MMP-10 (****P < 0.0001), and MMP-11 (****P < 0.0001), exhibited consistent repression, highlighting a broad regulatory footprint uncommon among anticancer interventions. The magnitude of suppression exhibited a clear treatment time dependency, with prolonged plasma exposure (240 s) producing stronger inhibitory effects than shorter treatments (120 and 180 s). Helium-driven DTAPPJ consistently induced a greater reduction in MMPs transcript levels than argon, highlighting the superior biological efficacy of helium-operated DTAPPJ (120, 180, 240 s; Fig. 4, Fig. 5, Fig. 6). The superior performance of helium-driven DTAPPJ compared to argon in all experiments can be attributed to fundamental plasma physics rather than the chemical properties of helium. Helium plasmas possess higher electron temperatures and mobility, which promote more efficient ionization and excitation, resulting in longer plasma plumes and improved propagation through the surrounding gas [103,104]. Furthermore, the lower breakdown voltage of helium allows plasma ignition and maintenance at reduced applied voltages, minimizing energy losses and ensuring more uniform delivery to the target [105,106]. Collectively, these physical characteristics enhance both the generation and transport of RONSs over extended distances, increasing their interaction with BCSCs and leading to more effective suppression of MMP expression. These plasma physics principles explain the consistent superiority of helium-driven DTAPPJ over argon-driven DTAPPJ across all exposure durations.Fig. 4. Analysis of MMP-1 expression following DTAPPJ exposure using (a) argon, (b) helium, and (c) a direct comparison between the two gas types (***P = 0.0006). Assessment of MMP-2 expression under DTAPPJ treatment using (d) argon, (e) helium, and (f) a comparative view of both gases (**P = 0.0011). Assessment of MMP-3 expression after DTAPPJ application using (g) argon, (h) helium, and (i) a side-by-side comparison of argon-versus helium-based treatment (***P = 0.0005).Fig. 4. Fig. 5Analysis of MMP-7 expression following DTAPPJ exposure using (a) argon, (b) helium, and (c) a direct comparison between the two gas types (***P = 0.0003). Assessment of MMP-9 expression under DTAPPJ treatment using (d) argon, (e) helium, and (f) a comparative view of both gases (***P = 0.0005). Assessment of MMP-10 expression after DTAPPJ application using (g) argon, (h) helium, and (i) a side-by-side comparison of argon-versus helium-based treatment (****P < 0.0001).Fig. 5. Fig. 6Analysis of MMP-11 expression following DTAPPJ exposure using (a) argon, (b) helium, and (c) a direct comparison between the two gas types (****P < 0.0001). Assessment of MMP-13 expression under DTAPPJ treatment using (d) argon, (e) helium, and (f) a comparative view of both gases (***P = 0.0002). Assessment of MMP-14 expression after DTAPPJ application using (g) argon, (h) helium, and (i) a side-by-side comparison of argon-versus helium-based treatment (**P = 0.0016).Fig. 6

These reactive species sustain oxidative pressure in 3D culture systems, driving transcriptional downregulation of multiple MMPs and reducing invasive potential [107,108]. Taken together, these findings demonstrate that DTAPPJ effectively suppresses a broad spectrum of MMPs involved in ECM remodeling, tumor invasion, and metastatic potential, likely through coordinated modulation of redox-sensitive transcription factors such as NF-κB, AP-1, STAT3, SP1, and HIF-1α [[109], [110], [111], [112]]. The uniformity of MMP repression suggests that DTAPPJ generates sufficient oxidative stress to trigger global transcriptional reprogramming in BCSCs, rather than isolated gene-specific effects, a mechanism particularly relevant given BCSCs’ reliance on MMP-mediated invasion and microenvironmental interactions to maintain stemness [113,114].

DTAPPJ-mediated MMP repression translated into pronounced reductions in BCSC metabolic activity (Fig. 7). Both argon and helium treatments induced dose-dependent viability loss, with a 240 s helium exposure producing the largest effect. This indicates that the oxidative load delivered by DTAPPJ can surpass the enhanced antioxidant defenses of BCSCs, which include elevated glutathione, catalase, and superoxide dismutase activity [115,116]. Previous studies of CAP cytotoxicity demonstrate mitochondrial depolarization, lipid peroxidation, DNA double-strand breaks, and caspase activation as central mechanisms [117,118]. By applying DTAPPJ within a 3D Matrigel system, we confirmed that these effects persist under physiologically relevant ECM constraints, which normally reinforce stemness, integrin–FAK–SRC signaling, and MMP expression [119,120]. This demonstrates that DTAPPJ effectively overrides microenvironmental reinforcement of invasive phenotypes. The low thermal footprint of DTAPPJ ensures that observed biological effects arise from chemical, not hyperthermic, mechanisms, consistent with prior CAP selectivity studies [121]. This feature, combined with the dual-transfer capability and low power operation, supports the feasibility of minimally invasive clinical applications, including endoscopic or intraluminal treatments where line-of-sight and thermal constraints limit conventional CAP usage.Fig. 7. Assessment of BCSC viability following DTAPPJ treatment: (a) argon, (b) helium, and (c) direct comparison between argon- and helium-based plasma.Fig. 7

Finally, the extent of suppression of the studied MMPs under the optimal DTAPPJ treatment parameters was measured and quantified (Fig. 8). The trend observed in Fig. 8, showing the hierarchy of MMP transcriptional inhibition under the most effective plasma condition (helium, 240 s), reflects the differential sensitivity of individual MMPs to DTAPPJ-mediated oxidative stress. Collagenolytic and invasion-associated MMPs such as MMP-3, MMP-9, and MMP-2 exhibited the strongest suppression, likely because their transcriptional regulation is highly responsive to intracellular redox perturbations induced by RONSs. In contrast, other MMPs (e.g., MMP-11, MMP-13) showed lower sensitivity, consistent with their distinct regulatory mechanisms and lower redox responsiveness. This pattern highlights that DTAPPJ selectively targets MMPs most critical for extracellular matrix degradation and metastatic potential, explaining the observed transcriptional trend. These results underscore both the anti-metastatic potential of DTAPPJ and the potential utility of these MMPs as biomarkers to monitor plasma efficacy in future translational studies.Fig. 8. Effects of DTAPPJ on the parameters evaluated in this investigation.Fig. 8

Collectively, the results from Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8 demonstrate that DTAPPJ provides long-range, low-power delivery of biologically active RONS, robust suppression of metastasis-associated proteases, and dose-dependent cytotoxicity in BCSCs. The dual-transfer architecture, with embedded copper wires acting as floating electrodes, ensures stable plasma propagation, enhanced reactive species generation, and safe operation with minimal thermal and electrical risks. Helium carrier gas maximizes reactive output and biological efficacy, making DTAPPJ a multi-targeted modality suitable for translational and potentially minimally invasive anticancer applications.

Conclusion

4

The DTAPPJ represents a significant advancement in cold plasma oncology, combining long-range, low-power delivery with efficient generation of biologically active RONSs. Helium-fed DTAPPJ demonstrated superior plasma stability, propagation, and reactive capacity, maintaining rich chemical profiles even at distal points within the dual-transfer conduit. Functionally, DTAPPJ exposure induced broad and coordinated transcriptional downregulation of multiple matrix metalloproteinases (MMP-1, -2, -3, -7, -9, -10, −11, −13, and −14), with the strongest suppression observed in MMP-14, MMP-2, and MMP-9. These molecular effects translated into significant, dose-dependent reductions in BCSC viability and metabolic activity, including within 3D tumor-like microenvironments, highlighting the platform's ability to overcome intrinsic antioxidant defenses and ECM-mediated reinforcement of stemness and invasiveness. Importantly, DTAPPJ achieves these effects through chemical mechanisms mediated by RONS rather than thermal damage, and the dual-transfer architecture ensures safe, stable plasma delivery with minimal electrical or thermal risks. Collectively, these findings establish DTAPPJ as a mechanistically coherent, technologically sophisticated, and biologically potent modality for targeting metastatic BCSCs. Future studies should focus on in vivo efficacy, detailed mapping of redox signaling pathways, and optimization of RONS flux to advance translational implementation in breast cancer therapy, supporting its potential as a minimally invasive anti-metastatic intervention.

CRediT authorship contribution statement

Abolfazl Soulat: Conceptualization, Methodology, Software, Writing – original draft, Writing – review & editing. Taghi Mohsenpour: Conceptualization, Data curation, Project administration, Supervision, Writing – review & editing. Leila Roshangar: Formal analysis, Methodology, Visualization, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.