The human breast cancer cell line MDA-MB-231 and patient-derived organoids (PDOs) from patients with TNBC were used as in-vitro tumor models, the human monocyte cell line THP-1 (kindly provided by Prof. Dr. Schenke-Layland Lab at the Department of Medical Technologies and Regenerative Medicine, at the University of Tübingen) as cellular immune compound, respectively. Four distinct culture media—M1, M2, M3, and M4—were utilized in these experiments. M1 consisted of Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Paisley, UK) supplemented with 10% fetal calf serum (FCS) and 1% penicillin-streptomycin (Gibco). M2 comprised DMEM without additional supplements, whereas M3 referred to RPMI 1640 GlutaMAX™ medium supplemented with 10% FCS and 1% penicillin-streptomycin. M4, specifically used for organoid cultivation, consisted of Advanced DMEM/F-12 supplemented with 1× GlutaMAX™ (Gibco), 10 mM HEPES (Gibco), and 1% penicillin-streptomycin (Gibco). M1 and M3 media were used for the seeding and cultivation of MDA-MB-231 and THP-1 cells, respectively. Cells were maintained in a humidified incubator at 37°C with 5% CO₂. The culture medium was refreshed every 2–4 days, and cells reaching 70–80% confluence were detached using 0.05% trypsin-EDTA (Gibco) for passaging. Organoids were derived from the pleural effusion of a patient with metastatic TNBC treated at the University Women’s Hospital in Tübingen, following written informed consent ( Table 1 ). The use of human donor cells was approved by the ethics committee of the medical faculty at the Eberhard Karl’s University Tübingen (495/2018BO2). Organoids were generated and cultured as previously described (25). Briefly, they were embedded in Basement Membrane Extract (BME, Bio-Techne) as single cells and seeded in 48-well plates at a density of 15,000 cells per well. Each well contained a 20 µL cell suspension-BME droplet at a ratio of 30% cell suspension to 70% BME. For passaging, organoids were washed and harvested in cold PBS/Y (PBS supplemented with 10 µM ROCK inhibitor Y-27632). Then, they were centrifuged, resuspended in TrypLE (Thermo Scientific) and incubated at 37°C in a water bath for 5 minutes. Following another centrifugation step, the supernatant was removed, and the pellet was resuspended in M4 medium for either reseeding or cryopreservation.

THP-1 cells were seeded in a 6-well plate at a density of 2 × 10⁶ cells per well and incubated in M3 medium with 80 nM phorbol 12-myristate 13-acetate (PMA; Sigma) for 48 hours to induce differentiation. This was followed by an additional 48-hour resting period in M3 medium without PMA to allow for macrophage maturation. The resulting THP-1-derived macrophages (M0) were then polarized by incubation with conditioned media for 24 hours. The conditioned medium was prepared as follows: MDA-MB-231 cells were treated with plasma, either APD or PTS, in the presence or absence of the STING inhibitor H-151, alongside a control group. Six hours after treatment, the existing medium was replaced with fresh M1 medium, and the cells were further cultured for an additional 48 hours. The supernatants from these five conditions were then collected and used as conditioned media for subsequent experiments.

The electrosurgical device VIO^® 3/APC 3 (Erbe Elektromedizin, Tübingen, Germany) was used to generate NIPP/APD in specifically tailored settings (Argon gas flow: 1.6 L/min; precise APC mode, effect 1). This mode delivers a continuous low-energy output, with an automatic precise adjustment of effect within a probe-to-tissue distance of up to 5 mm. For direct treatment, the previous medium was replaced by M2 medium. Cells covered with M2 medium were directly exposed to NIPP. For indirect treatment (PTS), M2 medium was firstly added into empty well and exposed to NIPP. Subsequently, the plasma-treated M2 medium (PTS) was transferred into the wells containing the pre-seeded cells, completely replacing the previous medium with PTS.

The concentration of RONS was measured using customary semiquantitative test strips “Quantofix Peroxide 25”, “Quantofix Peroxide 100” and “Quantofix Nitrat/Nitrit 100” (Macherey-Nagel) respectively. The colorimetric test strips adapt to a certain coloring in the presence of specific reactive species. For measurements, 1 mL of DMEM without supplements was exposed to NIPP for indicated energy, followed by immediate immersion of the test strips for 1 second and read out using QUANTOFIX Relax (Macherey-Nagel), which provided numerical values based on internal calibration.

Cell viability of MDA-MB-231 cells was assessed using the colorimetric MTS assay. Cells were seeded in a 24-well plate at a density of 8 × 10⁴ cells per well and incubated for 24 hours. Following plasma treatment, the total volume of the culture medium was adjusted to 500 µL for further incubation. After the designated incubation period, the medium was replaced with 300 µL of M1 medium supplemented with 30 µL of MTS reagent. The plates were then incubated for 2 hours. The absorbance was measured at 490 nm using a Varioskan™ LUX multimode microplate reader (Thermo Scientific).

Organoid cell viability was assessed using the CellTiter-Glo^® 3D Cell Viability Assay. Organoids were cultured for 11 to 14 days and subsequently treated with PTS. On the second day post-treatment, the culture medium was removed, and 200 µL of fresh M1 medium along with 200 µL of CellTiter-Glo^® reagent was added to each well. The contents were vigorously mixed for 5 minutes and then incubated at room temperature for 25 minutes. Luminescence was measured using Varioskan™ LUX multimode microplate reader (Thermo Scientific) with an integration time of 1 second per well.

MDA-MB-231 cells were seeded in a 24-well plate and incubated in M1 medium overnight. The following day, cells were subjected to gas flow (negative control), plasma treatment. After 6 hours of incubation, the culture medium was replaced with 200 µL of M1 medium and 200 µL of 2X RealTime-Glo™ Annexin V Apoptosis Detection Reagent (Promega). Apoptosis was measured every 8 hours for a total of 48 hours. Time point 0 referred to the first apoptosis measurement, taken 6 hours after plasma treatment. The detection reagent was prepared according to the manufacturer’s instructions by sequentially adding 1,000X Annexin NanoBiT^® Substrate, 1,000X CaCl₂, 1,000X Annexin V-SmBiT, and 1,000X Annexin V-LgBiT into prewarmed M1 medium. Luminescence was measured with an integration time of 1 second per well.

Proteins were extracted from tumor cells using RIPA lysis buffer (Thermo Fisher Scientific) mixed with protease inhibitors (MedChemExpress) and phosphatase inhibitors (Sigma Aldrich). Protein concentrations were quantified using the BCA protein assay kit (Thermo Fisher Scientific). Then, 100 µg of total protein was loaded per lane. Proteins were separated by 10% SDS-PAGE and transferred from gel to a nitrocellulose membrane (Sigma Aldrich) using Trans-Blot Turbo transfer system (Bio-Rad).

Membranes were blocked with 5% bovine serum albumin (BSA) in 0.1% TBST (0.1% Tween 20) and subsequently incubated overnight at 4°C with primary antibodies diluted in 0.1% TBST supplemented with 5% BSA. The following primary antibodies were used: anti-STING (#13647, 1:1,000 dilution, Cell Signaling Technology), anti-Phospho-STING (Ser366) (#19781, 1:1,000 dilution, Cell Signaling Technology), anti-Phospho-TBK1/NAK (Ser172) (#5483, 1:1,000 dilution, Cell Signaling Technology), anti-TBK1/NAK (#3504, 1:1,000 dilution, Cell Signaling Technology), anti-Phospho-Histone H2A.X (Ser139) (#9718, 1:1,000 dilution, Cell Signaling Technology), and anti-β-actin (#66009-1-Ig, 1:20,000 dilution, Proteintech).

On the next day, membranes were incubated for 1 hour at room temperature with anti-rabbit (#7074, 1:1,000 dilution, Signaling Technology) or anti-mouse (#7076, 1:1,000 dilution, Signaling Technology) secondary antibody conjugated to horseradish peroxidase diluted in 0.1% TBST supplemented with 5% BSA. Immunoblots were visualized using the ECL Western Blotting Substrate (Thermo Fisher Scientific) and imaged with the iBright CL1000 imaging system (Invitrogen).

Total RNA was extracted using 1 mL of TRIzol reagent (Invitrogen) per sample, following the manufacturer’s instructions. Complementary DNA (cDNA) synthesis was performed using M-MLV Reverse Transcriptase (Promega) according to the manufacturer’s protocol, utilizing the T100 Thermal Cycler (Bio-Rad). Quantitative real-time PCR (qRT-PCR) was conducted using the QuantStudio™ 3 Real-Time PCR System (Applied Biosystems) with PowerUp SYBR Green Master Mix (Applied Biosystems) and 2.5 µL of cDNA was used for each 20 µL reaction system. The qPCR program was 95°C for 2 minutes, followed by 40 cycles of 95°C for 15 seconds, 59°C for 15 seconds and 72°C for 1 minute. GAPDH was used as an internal reference control. The 2^−ΔΔCT method was used to calculate the relative mRNA level.

The following primers were used:

Adherent macrophages were detached using 0.05% trypsin-EDTA. The trypsinization process was halted by adding of M3 medium. The harvested cells were washed with PBS and resuspended in ice-cold FACS buffer (autoMACS^® Rinsing Solution supplemented with 0.5% bovine serum albumin) at a concentration of 1 × 10⁷ cells/mL. A 100 µL aliquot of the cell suspension was transferred to a universal tube and incubated with 2 µL of antibody in the dark at 4°C for 10 minutes. The antibodies used included FITC-labeled anti-CD11b (#AB_2536479, 1:50 dilution, Miltenyi Biotec) and APD-labeled anti-CD80 (#AB_1283666, 1:50 dilution, Miltenyi Biotec). Flow cytometric analysis was performed using a FACSCanto™ II flow cytometer, and data were analyzed with FlowJo 10.8.1 software (Becton Dickinson).

SPSS statistical software was used for data analysis. Statistical analyses were performed using a one-way ANOVA or Student’s t-test. A p-value of < 0.05 was considered statistically significant. Values are presented as mean ± standard error of mean (SEM). Each experiment was performed in at least three independent replicates.

During plasma treatment, a variety of reactive molecules are generated, with RONS being the most significant due to their extensive biological effects. Results demonstrated that plasma treatment led to a linear increase in the concentrations of H₂O₂, NO₂ ^-, and NO₃ ^- with increasing energy, as shown by the calculated rates of production (0.54, 0.04, and 0.54, respectively) and strong correlations (R² values of 0.93, 0.94, and 0.87, respectively) ( Figures 1A–C ). This suggests that higher energy input results in increased RONS concentrations, which in turn amplify the biological effects on the target. The plasma device generates higher energy by extending the exposure time. By recording both the exposure time and the final energy, we observed a proportional increase in energy over time ( Figure 1D ).

To investigate the cytotoxic effect of plasma treatment, MDA-MB-231 cells were treated with either APD or PTS. During APD treatment, cells were covered with 500 µL of M2 medium ( Figure 1E ). Both APD and PTS treatments exhibited similar cytotoxic effects after both 6 and 24 hours of incubation ( Figure 1F ). There was no significant difference between APD and PTS, suggesting that excessive covering medium may have attenuated the direct plasma treatment effect. To address this, we reduced the volume of the covering medium. To assess whether gas flow alone has an effect on cell viability, MDA-MB-231 cells were exposed to 36 seconds of direct argon gas flow without plasma ignition. This exposure time was chosen as it was sufficient for the plasma device to generate 128 Joules of energy, enough to induce significant cell death ( Figure 1F ). Cell viability significantly decreased without covering medium ( Figure 1G ). As medium volume increased, viability recovery occurred, with no further effect when the volume exceeded 300 µL.

To compare the cytotoxic effects of APD and PTS with decreasing medium volume, various volumes of M2 medium (100 µL to 500 µL) were used, all treated with 32 Joules of plasma energy. After treatment, the medium was adjusted to 500 µL. Results showed that under the same energy conditions, APD’s efficacy increased as covering medium volume decreased (p < 0.05), while PTS effect remained stable from 500 µL to 100 µL ( Figure 2A ). The reduction in medium volume helped minimize the neutralizing effect of the covering medium and excluded the influence of gas flow. Results showed that APD was more effective than PTS in decreasing the cell viability ( Figure 2B ). Apoptosis analysis revealed that both APD and PTS induced apoptosis, with APD showing an early onset of apoptosis at the 0-hour time point after treatment ( Figures 2C–F ).

We next investigated the impact of APD and PTS on the activation of the STING pathway at both the gene and protein expression levels. Since many therapeutics activate STING by inducing DNA damage, we first examined γ-H2AX, a marker of DNA double-strand breaks. Western blot analysis showed a significant increase in γ-H2AX expression following both APD and PTS treatments compared to the control group ( Figures 3A, B ). These aberrant DNA fragments are subsequently recognized by cGAS, which catalyzes the synthesis of 2′3′-cGAMP. This cyclic dinucleotide acts as a secondary messenger, binding to STING and initiating the signaling pathway (20). Western blotting showed an enhanced STING activation, as indicated by a higher p-STING/STING ratio in both APD- and PTS-treated cells ( Figures 3A, D ). As a key downstream effector of the STING pathway, p-TBK1 expression was further analyzed by western blotting in MDA-MB-231 cells following both direct and indirect plasma treatments. The results revealed a significant increase in p-TBK1 levels, while total TBK1 expression remained unchanged, leading to a markedly higher p-TBK1/TBK1 ratio compared to the control (p < 0.05) ( Figures 3A, E ).

To assess the persistence of these effects, we further examined protein expression at 24 hours post-treatment. Both direct (APD) and indirect (PTS) plasma treatments continued to induce DNA damage and sustained activation of the STING-TBK1 axis as shown by the increased expression level of γ-H2AX, as well as the p-STING/STING ratio and p-TBK1/TBK1 ratio at this timepoint ( Figures 3F–J ). After prolonged incubation, STING expression decreased in APD- and PTS-treated cells (p < 0.05), suggesting its degradation following signal transduction, consistent with findings from previous studies (26, 27) ( Figures 3C, H ).

The p-TBK1 protein is an important modulator for the IFN-β production. As a downstream event of STING activation, the transcription of IFN-β plays a crucial role in immune signaling. To further investigate the IFN-β signal pathway activation by APD and PTS, we used RT-PCR to determine the expression level of genes of IFN-β in MAD-MB-231 cells. The results revealed a significant upregulation of IFN-β expression following APD and PTS treatments ( Figure 3K ). Furthermore, key ISGs, including CCL5, CXCL10, IFIT1, ISG15, and OAS1, were also upregulated, indicating an immune response induced by plasma treatment ( Figure 3L ).

Taken together, plasma treatment effectively induced DNA double-strand breaks and subsequent STING pathway activation.

Within the tumor microenvironment, cancer cells exist in a complex network of interactions with various cell types, including fibroblasts, macrophages, and lymphocytes. To investigate how plasma-treated cancer cells influence surrounding immune cells and determine whether this effect is mediated through the STING pathway, we utilized the monocyte cell line THP-1. These cells were differentiated into M0 macrophages by incubation with 80 nM PMA for 48 hours ( Figure 4A ), a concentration and duration previously demonstrated to be effective (28). Morphologically, THP-1 cells appeared as round cells in suspension, while differentiated into M0 macrophages the adherent cells showed a round and partly donut-like shape. In contrast, after co-culture with conditioned media, activated macrophages exhibited an M1 phenotype, characterized by an elongated, amoeboid shape and numerous fibrillary cytoplasmic structures ( Figure 4B ).

To determine whether plasma-induced M1 polarization was STING-dependent, we employed the STING inhibitor H-151, which blocks STING activation by preventing its palmitoylation at the Golgi apparatus, thereby suppressing TBK1 activation (29). Western blot analysis revealed a reduction in p-TBK1 levels in APD- and PTS-treated samples when H-151 was present, confirming successful STING inhibition ( Figure 4C ). Flow cytometry analysis further demonstrated a significant increase in the percentage of CD11b⁺CD80⁺ M1 macrophages in the APD and PTS treatment groups compared to the control ( Figures 4D, E ). Notably, the addition of H-151 to MDA-MB-231 cells prior to PTS treatment significantly reduced this M1 polarization effect (p < 0.05). Furthermore, a numerical reduction was observed in the APD + H-151 group compared to the APD-only group.

Following the demonstration of plasma’s immunomodulatory effects using a commercially available breast cancer cell line, we employed patient-derived primary breast cancer organoids to further validate these findings, as organoids better mimic the physiological relevance of actual tumors compared to cell lines. The organoids were generated from pleural effusion obtained from a female patient with triple-negative breast cancer. After 10 to 14 days of incubation, the organoids formed dense and irregular structures ( Figure 5A ). The present data show that PTS induced M1 polarization in a STING-dependent manner ( Figure 4E ). Additionally, PTS was more manageable compared to APD in small wells. Once the organoid cultures were successfully established, plasma treatment effects were evaluated using PTS. Brightfield imaging revealed structural disruption of the organoids and extensive cell death ( Figure 5B ). Additionally, viability assays confirmed a dose-dependent reduction in organoid viability following PTS treatment ( Figure 5C ). The polarization of THP-1-derived macrophages was induced by transferring conditioned medium from the organoids, following the same protocol as shown in Figure 4A . Since the basement membrane extract (BME), used as a scaffold for organoid culture, is composed of biomaterial, it may have potential effects on M1 polarization when exposed to plasma. To account for this, an additional control group containing only the BME scaffold (without cells) was included. After plasma treatment, the mean ratio of CD11b⁺CD80⁺/CD11b⁺ cells increased, indicating M1 polarization. Importantly, this increase was not attributed to the plasma-treated BME alone ( Figures 5D, E ).