Pleiotropic Devitalization of Renal Cancer Cells by Non-Invasive Physical Plasma: Characterization of Molecular and Cellular Efficacy

Simple Summary Renal cell carcinoma (RCC) has an extremely poor prognosis, as there are few therapeutic options. In addition, resistance often develops during the administration of cytostatic drugs, preventing further drug-based therapy. New therapeutic modalities to reduce tumor cells would therefore be an important advance in the cancer therapy of RCC. In the present research study, non-invasive physical plasma (NIPP) was used to treat RCC cells. It was found that both cell growth and cell motility of RCC cells could be significantly reduced. Furthermore, programmed cell death was induced, so that some of the cancer cells actually died. Cellular analysis of NIPP treated cancer cells indicated that exposure to NIPP resulted in increased permeability of the cytoplasmic membrane to low molecular weight substances. This may contribute to the inhibition of the physiological activities of the cancer cells. The present results suggest that treatment with NIPP may represent a promising, innovative, and non-chemical option for the therapy of RCC and cancer in general. In combination with established therapeutic modalities, therapeutic effects would be enhanced and resistance would be minimized by the simultaneous application of multiple modes of action. Abstract Renal cell carcinoma (RCC) is the third most common urological tumor and has an extremely poor prognosis after metastasis has occurred. Therapeutic options are highly restricted, primarily due to resistance to classical chemotherapeutics. The development of new, innovative therapeutic procedures is thus of great urgency. In the present study, the influence of non-invasive physical plasma (NIPP) on malignant and non-malignant renal cells is characterized. The biological efficacy of NIPP has been demonstrated in malignant renal cell lines (786-O, Caki-1) and non-malignant primary human renal epithelial cells (HREpC). The cell responses that were experimentally examined were cell growth (cell number determination, calculation of growth rate and doubling time), cell motility (scratch assay, invasiveness assay), membrane integrity (uptake of fluorescent dye, ATP release), and induction of apoptosis (TUNEL assay, caspase-3/7 assay, comet assay). A single NIPP treatment of the malignant cells significantly inhibited cell proliferation, invasiveness, and metastasis. This treatment has been attributed to the disruption of membrane functionality and the induction of apoptotic mechanisms. Comparison of NIPP sensitivity of malignant 786-O and Caki-1 cells with non-malignant HREpC cells showed significant differences. Our results suggest that renal cancer cells are significantly more sensitive to NIPP than non-malignant renal cells. Treatment with NIPP could represent a promising innovative option for the therapy of RCC and might supplement established treatment procedures. Of high clinical relevance would be the chemo-sensitizing properties of NIPP, which could potentially allow a combination of NIPP treatment with low-dose chemotherapy.

Tobacco consumption, obesity, and hypertension are among the most prominent factors for RCC development [7]. Despite novel abdominal imaging procedures, more than 25% of RCC patients are initially diagnosed with metastases [8].
The current therapy concepts depend on the tumor stage and the patient's health condition. Focal therapies, e.g., cryofrequency ablation, are used to treat RCC patients with pronounced comorbidities or limited life expectancy [9][10][11][12]. In curative therapy, surgical resection of the tumor plays a pivotal role [13]. Kidney-preserving operations and partial nephrectomy can be performed on small, localized tumors [9,12,13]. Total nephrectomy in the palliative situation facilitates the reduction in the tumor burden [14,15].
Systemic antitumor therapy of RCC is only available to a very limited extent since there are frequently pronounced resistances to classical chemotherapeutic agents [16,17] and a lack of responsiveness to immunotherapeutic agents such as interferon-α or interleukin-2 [18,19]. The currently available targeted therapy approaches with tyrosine kinase and mTOR inhibitors are significantly more efficient and have thus replaced the classic therapy concepts mentioned above [9]. Targeted therapy prolongs overall survival from 21.8 to 26.4 months [20,21] with a 5 year survival rate of about 75%, depending on the tumor stage [5,22].
As with almost all malignancies, the molecular heterogeneity of RCC makes effective therapy difficult [23][24][25][26]. New therapeutic strategies are therefore urgently needed to improve the current treatment options. One option may be treatment with non-thermal physical plasma at atmospheric pressure (non-invasive physical plasma: NIPP) [27,28]. Various studies have shown the antiproliferative effect of NIPP on cancer cells of melanoma, breast, ovary, and lung, as well as skeletal sarcoma [29][30][31][32][33][34][35]. The antiproliferative effect of NIPP is based on the induction of apoptosis due to the production of reactive oxygen (ROS) and nitrogen species (RNS) [36] and limitations of cellular metabolism and membrane functionality [35,37,38]. The combination of NIPP treatment with classical chemotherapeutic agents also appears promising and could overcome existing chemoresistance [39]. To our best knowledge, the present investigation represents the first study on antitumoral effects of NIPP on RCC cells

NIPP Treatment Inhibits Cell Proliferation by Reducing Cell Number and Growth Rate
The malignant cell lines demonstrated inhibition of cellular growth after NIPP treatment ( Figure 1). While in Caki-1 cells, a 5 s treatment resulted in a significant reduction in the cell number, only slight differences were observed in 786-O cells. From treatment times of 10 s, however, the proliferation of the malignant cell lines was significantly inhibited and reduced the number of living cells to about half after 120 h of incubation. Finally, a 20 s treatment led to almost complete growth inhibition in malignant cell lines. In contrast, the growth of the non-malignant cells was not significantly inhibited. However, it was observed that the primary cells (HREpC) began to die after 96 h regardless of treatment.
Biostatistical analyses showed that these effects were due to the reduction in the number of cells during treatment and to a reduction in the growth rate of surviving cells (Table 1). Caki-1 (D-F) and the non-malignant HREpC cells (G-I), were treated with non-invasive physical plasma (NIPP) or the carrier gas argon (Ctrl). The number of living cells was determined at the given times using the CASY Cell Counter and Analyzer and were shown as mean ± SD. Differences between NIPP-treated cells and control cells were tested for significance using the paired t-test (* p < 0.05, ** p ≤ 0.01, *** p ≤ 0.001). For a better overview, the values of the NIPP treatment were shown slightly shifted to the right.
The calculated cell numbers of 786-O cells immediately after treatment differed significantly after NIPP treatments over 10 s and 20 s compared to controls (each p < 0.001). A comparison of the growth rates of 786-O cells demonstrated that the growth rate of the 20 s NIPP-treated cells was significantly reduced (p < 0.001). In contrast, NIPP treatment over 5 s and 10 s tended to result in a discrete increase in growth rate. After NIPP treatment of Caki-1 cells over 10 s and 20 s, the calculated cell numbers at time t = 0 (each p < 0.001) and the growth rates (each p < 0.001) decreased significantly. The treatment of HREpC showed no significant effects. The generation of NIPP in atmospheric environment is associated with the formation of reactive oxygen species (ROS). To confirm this in the experimental setting, the cell culture media used, RPMI, MEM, and RCM, were treated with NIPP. The formation of H 2 O 2 as a major representative of ROS was measured using an Amplex Red assay ( Figure 2). The concentration of H 2 O 2 formed increased with increasing NIPP exposure time in all cell culture media tested. Figure 2. RPMI, MEM, and RCM cell culture media were treated with different NIPP treatment times and the formation of H 2 O 2 was measured by Amplex Red assay. Data were expressed as mean ± SD. Significant differences between NIPP treatment and untreated controls (0 s; control = 1.0) are shown as follows: * p < 0.05, ** p < 0.001, **** p < 0.0001.

Incubation of Untreated Cells with NIPP-Treated Cell Culture Medium also Results in Cell Growth Inhibition
The experiments mentioned above demonstrated the growth inhibitory efficacy of NIPP treatment. We now examined whether NIPP effects can also be mediated indirectly through NIPP-treated cell culture medium and whether this also leads to reduced prolifera-tion. For this purpose, the cells were seeded before treatment and pre-incubated 24 h. After preincubation, the cell culture medium was replaced by NIPP-or argon-treated medium and incubated for another 120 h. Treatment with a NIPP-activated medium inhibited cell proliferation in all cell lines ( Figure 3). activated cell culture media. After 24 h of pre-incubation, the cell culture medium was replaced by NIPP or argon-treated medium and the cells were incubated for further 120 h. The number of living cells was determined at the given timepoints by using the CASY Cell Counter and Analyzer and displayed as mean ± SD. Differences between the NIPP-activated media-treated cells and the control cells were tested for significance using the paired t-test (* p < 0.05, ** p ≤ 0.01, *** p ≤ 0.001). For clarity, the values of the NIPP treatment were shown slightly shifted to the right. Treatment with 5 s NIPP-activated medium resulted in a slight growth inhibition. Only 768-O cells showed significant differences at two points in incubation (24 h: p = 0.018, 48 h: p = 0.049). A 10 s treatment of cell culture medium resulted in approximately half-maximal inhibition in the malignant cell lines. Significant differences were found at all points from 48 h (p < 0.05). Cell proliferation was almost completely prevented in a NIPP-treated medium for more than 20 s. Here, significant differences were also found from 48 h on (p < 0.05). In contrast, the growth of the non-malignant cells (HREpC) was not significantly inhibited ( Table 2). Significant difference (p < 0.05) between non-invasive physical plasma (NIPP) activated media-treated and the corresponding control (Ctrl) were marked in bold.
Incubation of 786-O cells with NIPP-treated medium also led to a reduction in the number of cells. A significant difference was observed when incubated with the 20 s treated medium (p = 0.046). Indirect treatments of 786-O cells with NIPP activated medium for 10 s and 20 s led to a significant reduction in the growth rate (10 s: p = 0.004, 20 s: p < 0.001).
Indirect treatment of Caki-1 cells also reduced the number of cells at time t = 0. Significant differences were found at 10 s (p = 0.013) and 20 s (p = 0.012) treatment duration. For Caki-1 cells, the effects of indirect treatment were less pronounced. Here, there was a tendency for 5 s and 10 s NIPP treatment to increase the growth rate.
Indirect treatment of HREpC cells did not result in significantly different cell counts at time t = 0 or the growth rate.

Treatment with NIPP Induces Apoptosis in Cells
Malignant cell lines 786 O, Caki-1, and non-malignant HREpC cells were used to examine whether treatment with NIPP leads to the activation of apoptotic processes. For this purpose, the established apoptosis detection methods TUNEL assay and Caspase 3/7 activation assay were performed. The cells were treated with NIPP or argon for 10 s. The TUNEL assay was performed 24 h and 48 h, the Caspase 3/7 assay 24 h, 48 h, and 72 h after NIPP treatment ( Figure 4).
The treatment of 786-O cells with NIPP increased the relative TUNEL signal per cell to 2.6-fold (p = 0.005) after 24 h and to 2.8-fold (p = 0.003) after 48 h. At the same time, the relative caspase 3/7 activity increased by NIPP treatment (24 h: 3.7-fold, p = 0.031; 48 h: 3.8-fold, p = 0.037; 72 h: 3.2-fold, p = 0.025). Caki-1 cells exhibited similar results. A significant increase in relative TUNEL signal was observed 24 h (1.9.-fold, p = 0.044) and 48 h (2.5-fold, p = 0.046) after NIPP treatment. The relative caspase 3/7 activity was also increased (24 h: 1.1-fold, p = 0.026, 48 h: 1.2-fold, p = 0.006, 72 h: 1.8-fold, p = 0.047). Apoptotic effects in the non-malignant HREpC cells were less pronounced. NIPP treatment did not significantly increase the relative TUNEL signal at any of the observed time points. Caspase 3/7 activity was slightly reduced compared to the argon control. Seventy-two hours after NIPP treatment, the caspase 3/7 activity was significantly decreased compared to the control (0.6-fold p = 0.006). non-malignant HREpC (E,F) were treated with non-invasive physical plasma (NIPP) or carrier gas argon and examined for apoptosis using the established apoptosis detection methods TUNEL assay (A,C,E) and Caspase 3/7 activation assay (B,D,F). Means ± SD of the relative signal intensity are shown normalized to the respective argon control. The data were tested for significant differences using a paired t-test (* p < 0.05, ** p ≤ 0.01). To validate the TUNEL assay, positive and negative controls were carried out (+ nuclease-pretreated positive control,-unlabeled negative control). Those positive and negative controls were normalized to the mean of the argon controls.

Treatment with NIPP Reduces Cell Motility and Invasiveness
Scratch assays were performed to investigate a potential impact of NIPP on cell motility. For this purpose, NIPP-and argon-treated cells were seeded and the confluent cell layer was scratched after 24 h. The cell layer was imaged hourly and the cell-free area was determined. An invasion assay was performed to explore the effect of NIPP on cell invasiveness. Cells were treated with NIPP and argon and seeded in chambers with a semi-permeable bottom membrane. An FCS gradient was established across the membrane and cells were incubated for 24 h. The adherent cells on the top and bottom of the membrane were counted ( Figure 5). . Cells were cultured with low-serum conditions. The cell free area was photographed every half hour and measured at the indicated times. The relative area to the starting area at time t = 0 was evaluated. Migration assay (C,D): Treated cells were seeded in chambers with a semi-permeable membrane. An FCS gradient was established over the membrane. After 24 h of incubation, the cells on the top and bottom of the membrane were counted, and the invaded cells were compared to the total number of cells. Means ± SD are shown. Differences between the NIPP-treated cells and the control cells were tested for significance by students t-test (* p < 0.05, ** p ≤ 0.01).
Cell motility was cell line specific. The cell-free area of 786-O cells was almost completely closed after just a few hours. In contrast, Caki-1 cells could not completely close the free area for 48 h. The influence of motility through NIPP treatment also depended on the cell line. At any time of the evaluated measurement times, no significant differences were observed in 786-O cells. Significant differences, however, were found in Caki-1 cells after 8 h. HREpC cells died under low-serum conditions.
The cell lines also differed in their invasiveness. While more than 80% of the controltreated 786-O cells were invasive, only slightly than half of the Caki-1 cells were invasive. NIPP treatment significantly reduced the invasiveness of the 786-O cells. While 84% ± 6% of the control-treated cells invaded through the membrane, the NIPP-treated cells were only 27% ± 19% (p = 0.008). The NIPP impact was less obvious in Caki-1 cells; however, the results also indicated a reduction in the invasiveness in this cell line. While 55% ± 16% migrated from the control cells, this proportion decreased to 35% ± 11% (p = 0.149) after NIPP treatment. HREpC cells died under low-serum conditions.

Treatment with NIPP Impairs the Functionality of the Cytoplasmic Membrane
The generation of reactive species in the close environment and on the surface of the cells suggests that the efficacy of NIPP might alter the functionality of the cytoplasmic membrane. To investigate the integrity of the cytoplasmic membrane, cells were stained with FDA and treated with NIPP and argon. After an incubation of 15 min, cells were sedimented and the dye content of the cells was determined ( Figure 6A-C). An ATP release assay was performed to investigate membrane permeability further. In contrast to the dye-based method, cells were not incubated with low-molecular-weight substances, but the release of an endogenously synthesized biomolecule was measured ( Figure 6D-F). Dose-dependent differences were also observed in Caki-1 cells; however, these were not significant at any of the treatment times investigated. HREpC cells displayed no significant differences between NIPP and control.

Discussion
NIPP's cell growth-inhibitory effects have been proven in numerous studies with different types of malignant cells [38,[40][41][42][43]. NIPP is a multi-component system consisting of ROS and other charged and uncharged particles, electric fields, and UV light. NIPPderived ROS appear to play a central role as therapeutic pathways in the therapy of various malignancies [44,45]. It appears that the cell non-autonomous mechanisms manifest themselves biologically as a physical effect of the components of the NIPP. There is evidence that NIPP treatment of malignant cells leads to increased compaction of the cell membrane and thus to its biological protective function for the cell [35,46]. In addition, much is reported in the literature about the NIPP-induced apoptosis mechanisms stimulated in malignant cells [38,47,48].
In the field of uro-oncology, investigations on the application of NIPP in prostate and bladder tumors have already been carried out [49,50]. In our current study, the previously discussed effects of activation of apoptosis cascades and impairment of membrane permeability are demonstrated for the first time in malignant renal cells.
It was shown that a single NIPP treatment significantly inhibited cell growth of RCC cells. Furthermore, the strength of the antiproliferative effect increased with the duration of NIPP treatment, as was confirmed for other malignancies including ovarian, breast, and pancreatic cancer cells [32,38,51]. The primary noxious agent leading to this growth inhibition is reactive low-molecular-weight compounds, mainly reactive oxygen, and nitrogen species [37,52]. These are formed at the interfaces of NIPP and ambient atmosphere and of NIPP and liquid medium [53]. Therefore, the concentration of reactive species increases with continued treatment [36].
Reactive species as the predominant active component were also shown to inhibit cell growth not only in direct NIPP treatment. Growth inhibition also occurred after indirect treatment when untreated cells were incubated with NIPP-treated medium. In both cases, reactive species' highly elevated extra-and intracellular concentrations lead to cell cycle arrest and cell death [54]. Conversely, low intracellular concentrations of reactive species activate the cell cycle and induce increased cell proliferation [54,55]. These effects are used for example in NIPP therapy of chronic wounds [56]. The main difference between direct and indirect NIPP treatment is that direct treatment may also have biological effects due to electromagnetic and thermal radiation. In contrast, indirect treatment only enables reactive species to be transported through the liquid medium [29,57]. Therefore, the cell biological effects of indirect treatment often appear less pronounced [58]; however, the present examinations have shown that NIPP treatment is almost equally effective for malignant and non-malignant renal cells, which was also observed in other studies [37,52]. In addition, the experimental use of NIPP can also lead to chemical changes in cell culture medium components in both direct and indirect treatment. This would also interfere with cellular metabolism, e.g., by chemically modifying amino acids, and thus cause a decrease in cell growth [36,59].
Since NIPP treatment did affect not only cell growth but also other central physiological functions of the cells, it had to be assumed that the NIPP effect cannot be solely attributed to disturbances in metabolic processes. It also seemed that general signal and effector cascades were dysregulated by NIPP components. Membrane permeability experiments for fluorescence-labelled low-molecular substances demonstrated an increased permeability after NIPP treatment. Cellular ATP molecules could also cross through the cytoplasmic membrane into the extracellular space, which may have contributed to metabolically induced growth inhibition. Comparable studies confirm this in melanoma and pancre-atic carcinoma cells [60]. Immunological functions, however, cannot be excluded in this procedure. In apoptotic processes ATP may be secreted to activate macrophages [61,62].
Alterations in membrane functionality may also have contributed to NIPP-induced inhibition of cell motility. Membrane-bound adhesion proteins such as integrins and cadherins are central factors for cell contacts and cell mobility and be modulated by CPA treatment [56,63]. Treatment with NIPP can lead to chemical modifications of biomolecules, e.g. through oxidation processes. Therefore chemical modification of cellular surface factors such as lipids and proteins is not unlikely [64,65]. Inhibition of metastasis and invasiveness has already been shown for ovarian, colon, and hepatocellular carcinoma cells [32,66,67] and has also been demonstrated in RCC cells. The present study showed clear differences between the individual cell lines. The 786-O cells had a significantly higher metastatic and invasive potential than Caki-1 cells. The cell-free area in the scratch assay was closed six-fold faster by 786-O cells and the proportion of invasive cells was 80%, about one third higher than in the other two cell lines. This high cell mobility of 786-O cells was also demonstrated in other studies [68].
Another important cellular effect of NIPP treatment is the activation of the apoptotic machinery [69,70]. This has been shown at the activation level of the apoptotic proteases' caspase-3 and caspase-7, as well as the apoptosis-specific degradation of genomic DNA [71] This difference was also reflected in the apoptosis assays. Here the non-malignant cells showed a lower and later apoptotic response.
One hypothesis on the biological effect of NIPP suggests that cancer cells are significantly more affected by NIPP treatment than neighboring non-malignant cells [72]. This assumption is not uncontroversial and was also not supported by a recent study of us analyzing four cell lines after the treatment with three NIPP devices [73]. Although not the primary question, the present research was suitable for providing new insights into this hypothesis.
Cellular motility, invasiveness, and membrane permeability showed no significant differences between malignant and non-malignant cells after NIPP treatment. The question of a significantly stronger effect of NIPP on cancer cells still cannot be answered definitively. It is possible that permanent cell lines do not represent a suitable model system for these investigations due to their degenerated signaling networks and resulting unphysiological cell responses. The use of models with primary material might be able to remedy this situation.
The present studies on the biological effects of NIPP on renal cells show clear antiproliferative and antionogenic effects of NIPP treatment. These are mainly due to the induction of apoptosis and the disturbance of cytoplasmic membrane function. Treatment of tumors with NIPP remains a promising innovative option for new therapeutic methods in oncology. However, the effects of the different plasma devices with their different carrier gases must be adapted to the respective malignancy [37]. In the case of RCC, the combination of NIPP treatment and local chemotherapy could offer a promising new approach to treat this challenging malignancy.

Direct NIPP Treatment
A total of 1.0 × 10 4 cells were suspended in 200 µL medium and transferred to a 24-well cell culture plate. NIPP treatment was performed with the kIN-Pen®MED plasma jet (Neoplas tools, Greifswald, Germany). The flow rate of the carrier gas argon was adjusted to 3 slm. Control cells were treated analogously, but without inflaming the plasma flame. Cell suspensions were treated for 5 s, 10 s and 20 s. Immediately after the treatment, 800 µL medium each were added to the wells and cells were incubated for 120 h. The number of living cells was determined after 4 h, 24 h, 48 h, 72 h, 96 h, 120 h using CASY cell counter and analyzer model TT (OLS OMNI Life Science, Bremen, Germany).

Indirect NIPP Treatment
A total of 5.0 × 10 3 cells were seeded into the wells of a 24-well cell culture plate. After 24 h incubation, fresh preheated cell culture medium was activated with NIPP in another 24-well plate. Analogous to direct treatment, 200 µL of medium were treated per well. The treatment times corresponded to those of direct treatment. Immediately after treatment, the medium was carefully aspirated from the incubated cells and replaced by the treated medium. Afterwards, a further 800 µL of untreated medium was added to the wells. The cell number was determined and evaluated as described under Section 4.2.

Hydrogen Peroxide (H 2 O 2 ) Formation Assay
A total of 200 µL of RPMI 1640, MEM (both containing 2 mM L-glutamine, 1% P/S and 10% FCS) and RCM (containing SupplementMix) were treated with NIPP for 0, 5, 10, 30 and 60 s. The cell culture medias were treated analogously to the cell suspensions in our other experiments. The treated media was diluted at 1:50 and the Amplex Red hydrogen peroxide assay (Thermo Fisher Scientific, Waltham, MA, USA) was carried out according to the manufacturer's instructions. The background was subtracted. The data were analyzed relative to the control (control = 1.0).

Calculation of Growth Rate and the Nummber of Cell at the Time Immediatley after the Treatment
The growth rate and the number of cells at the time immediately after the treatment were calculated from the measured cell numbers after direct or indirect treatment using the following model: Y: cell count; Y0: cell count at t = 0; k: growth rate; t = time For easier comparability, the doubling time was calculated from the growth rate using the following formula: Doubling time = ln(2)/k Since the HNEpC began to die after 96 h, only the first 96 h after treatment were analyzed, in which the cells showed an exponential growth.

TUNEL-Assay
A total of 5.0 × 10 4 cells were treated with NIPP or argon for 10 s. 5.0 × 10 4 (24 h incubation) or 2.5 × 10 4 (48 h incubation) of the treated cells were transferred to a 96-well plate. Additionally, negative controls lacking fluorescent labelling and a nucleasetreated positive control were included. To be able to normalize the measured absorption to the cell number later, a second cell culture plate was simultaneously performed. The TiterTACS™ Colorimetric Apoptosis Detection Kit (Trevigen, Gaithersburg, MD, USA) was used according to the manufacturer's instructions. Absorption was measured using the Infinite M200 plate reader (Tecan, Männedorf, Switzerland).

Caspase-3/7-Assay
A total of 5.0 × 10 4 cells were treated with NIPP or argon for 10 s. 5 × 10 4 (24 h incubation), 2.5 × 10 4 (48 h incubation) or 1.25 × 10 4 (72 h incubation) of the treated cells were incubated for 24 h, 48 h or 72 h. To be able to normalize the measured fluorescence intensity to the cell number, a second plate was planted out parallel. After the incubation period, the used medium was removed and 100 µL of Caspase 3/7 detection solution (CellEvent TM Caspase 3/7 Green Detection Reagent (Thermo Fisher Scientific, Waltham, Massachusetts, USA) 2 µM in DPBS) were incubated for 45 min. The fluorescence (535 nm) was measured using a plate reader

Scratch-Assay
A total of 6.0 × 10 5 (786-O, or 5.0 × 10 5 (Caki-1) cells were seeded 24 h before the start of the assay. The confluent cell layer was scratched with a 250 µL pipette tip. Cells were washed twice with DPBS and 200 µL NIPP-or argon-treated medium wash to prevent the scratch from being closed by proliferation, the FCS concentrations in the medium were strongly reduced (786-O: 0.1% FCS, Caki-1: 0.5% FCS). By using the software Zen 2012 pro, the cell-free area was recorded over 48 h in intervals of 1 h. Evaluation of the cell-free area was determined with the ImageJ software using the MRI Wound Healing Tool plug-in. The measured cell-free areas were relativized to the cell-free area at the beginning of the experiment.

Invasion-Assay
To investigate the invasive capacity of the cells, invasion assays based on the Boyden chamber [74] were performed. For this purpose, cell culture inserts were inserted into the wells of a 24-well cell culture plate. The bottom of these inserts consisted of an opaque PET membrane with 8 µm pores. For the assay, 5.0 × 10 4 cells were suspended in 200 µL FCSfree medium and treated with NIPP or argon for 10 s. Treated cell suspensions were filled with 500 µL FCS-free medium and transferred to the cell culture inserts. The wells of the cell culture plate itself were filled with FCS-containing medium. After 24 h incubation, the medium was carefully aspirated, and the inserts removed. The membranes were removed from the insert. After washing twice in DPBS the cells were stained with DAPI (500 nM in DPBS) and applied to slides. The membranes were covered with coverslips and signals were measured on both sides of the membrane. With the BZ-II Analyzer software, the number of cells was determined. The number of penetrated cells was evaluated in relation to the total number of cells. The test setup is shown in Figure 7.

Loss of Dye Assay
Cells were harvested and diluted to 1.0 × 10 6 cells per milliliter in measuring buffer (Dulbecco's phosphate-buffered saline (DPBS) with 10% FCS (v/v)). Subsequently, the cell suspension was treated with NIPP or argon for 60 s. Immediately after, the treatment cells were stained with 30 µg/mL ethidium bromide and 5 µg/mL FDA and incubated for 15 min on ice in the dark. After centrifugation, the labelled cells were resuspended in measuring buffer and analyzed in a FACSCanto flow cytometer (BD Biosciences, Heidelberg, Germany) with the FACSDiva 6.0 Software (BD Biosciences) and evaluated with FlowJo Software Version 10 (Tree Star Inc., Ashland, OR, USA). The gating strategy is shown in Figure 8. The signals of the NIPP-treated cells were normalized to the signals of control cells.

ATP Release Assay
Cells were harvested and adjusted to 1.0 × 10 6 cells/ml with DPBS. A total of 200 µL of the cell suspension was treated with NIPPor argon for 5 s, 10 s, 30 s, or 60 s. After treatment, cells were incubated for 10 min at room temperature. After centrifugation, the relative ATP concentration of the cell-free supernatant was determined using the CellTiter-Glo 2.0 reagent (Promega GmbH, Walldorf, Germany) in an Infinite M200 plate reader.

Conclusions
We have shown that NIPP treatment effectively inhibits RCC cell proliferation. This effect was due to the impairment of cytoplasmic membrane and cell cycle functionality as well as the induction of apoptosis. These in vitro results give rise to the hope that NIPP treatment could also be used in vivo for the treatment of RCC including secondary tumors. If applicated intraoperatively, cells remaining in situ might be inhibited and their metas-tasis and invasiveness might be reduced. In the future, even endoscopic or laparoscopic NIPP application would be conceivable. An additional rinsing of the body cavity with NIPP-activated fluid, optionally in combination with low-dose chemotherapeutic agents, could further improve the prognosis. NIPP has the potential to significantly expand the opportunities for RCC therapy.