Inhibition of Angiogenesis by Treatment with Cold Atmospheric Plasma as a Promising Therapeutic Approach in Oncology

Background: Cold atmospheric plasma (CAP) is increasingly used in the field of oncology. Many of the mechanisms of action of CAP, such as inhibiting proliferation, DNA breakage, or the destruction of cell membrane integrity, have been investigated in many different types of tumors. In this regard, data are available from both in vivo and in vitro studies. Not only the direct treatment of a tumor but also the influence on its blood supply play a decisive role in the success of the therapy and the patient’s further prognosis. Whether the CAP influences this process is unknown, and the first indications in this regard are addressed in this study. Methods: Two different devices, kINPen and MiniJet, were used as CAP sources. Human endothelial cell line HDMEC were treated directly and indirectly with CAP, and growth kinetics were performed. To indicate apoptotic processes, caspase-3/7 assay and TUNEL assay were used. The influence of CAP on cellular metabolism was examined using the MTT and glucose assay. After CAP exposure, tube formation assay was performed to examine the capillary tube formation abilities of HDMEC and their migration was messured in separate assays. To investigate in a possible mutagenic effect of CAP treatment, a hypoxanthine-guanine-phosphoribosyl-transferase assay with non malignant cell (CCL-93) line was performed. Results: The direct CAP treatment of the HDMEC showed a robust growth-inhibiting effect, but the indirect one did not. The MMT assay showed an apparent reduction in cell metabolism in the first 24 h after CAP treatment, which appeared to normalize 48 h and 72 h after CAP application. These results were also confirmed by the glucose assay. The caspase 3/7 assay and TUNEL assay showed a significant increase in apoptotic processes in the HDMEC after CAP treatment. These results were independent of the CAP device. Both the migration and tube formation of HDMEC were significant inhibited after CAP-treatment. No malignant effects could be demonstrated by the CAP treatment on a non-malignant cell line.


Results
Human Dermal Microvascular Endothelial Cells (HDMEC) were directly treated with cold atmospheric plasma (CAP) and compared to cells treated with argon carrier gas only (mock control). Direct treatment of HDMEC was performed with the two different CAP devices: kINPen (neoplas tools, Greifswald, Germany) and Mini Jet (Heuermann HF-Technik GmbH). Anti-proliferative effects were observed with both CAP devices, which led to statistically significant growth inhibition from an incubation period of 48 h ( Figure 1A,B). Growth inhibitory effects were treatment time-dependent (data not shown). For both CAP devices, a treatment time was determined, in which the cell number of CAP-treated cells was reduced to approximately half that of the control group treated with argon. In the case of kINPen, this was achieved by 15 s treatment time. After 120 h, the cell number of CAP-treated cells was 50.3 ± 6.0% compared to the control group. Treatment time had to be increased for the Mini Jet to obtain an anti-proliferative effect comparable to the treatment with kINPen. This was achieved by a 30 s treatment time. After 120 h, the cell number of CAP-treated cells showed 49.8 ± 7.8% compared to the control group. These treatment times were applied in all subsequent experiments.
Direct treatment of HDMEC was performed with the two different CAP devices: kINPen (neoplas tools, Greifswald, Germany) and Mini Jet (Heuermann HF-Technik GmbH). Anti-proliferative effects were observed with both CAP devices, which led to statistically significant growth inhibition from an incubation period of 48 h ( Figure 1A,B). Growth inhibitory effects were treatment time-dependent (data not shown). For both CAP devices, a treatment time was determined, in which the cell number of CAP-treated cells was reduced to approximately half that of the control group treated with argon. In the case of kINPen, this was achieved by 15 s treatment time. After 120 h, the cell number of CAPtreated cells was 50.3 ± 6.0% compared to the control group. Treatment time had to be increased for the Mini Jet to obtain an anti-proliferative effect comparable to the treatment with kINPen. This was achieved by a 30 s treatment time. After 120 h, the cell number of CAP-treated cells showed 49.8 ± 7.8% compared to the control group. These treatment times were applied in all subsequent experiments.
The anti-proliferative effect of CAP treatment on cells could possibly be influenced by components of the cell culture medium. In particular, chemical modifications of these componentse.g., amino acids-could influence cellular metabolism. To investigate this indirect influence, untreated HDMEC were incubated with CAP-and argon-treated cell culture media for 120 h. The number of viable cells was examined 4,24,48,72,96, and 120 h after treatment. For both CAP devices, no statistically significant difference in growth between the indirectly treated HDMEC compared to control cells was found ( Figure 1C,D).  Curbing the HDMEC growth after direct and indirect CAP-treatment. HDMEC were treated with cold atmospheric plasma (CAP) generated by a kINPen (A) or Mini Jet (B) device. HDMEC were treated with CAP-treated cell culture media by kINPen (C) and by Mini Jet (D). As control (CTRL) cells were treated with the carrier gas argon. The number of viable cells was examined at the indicated times. Four independent experiments were performed. Results are presented as means ± SD. Two-way RM ANOVA was performed. Student's t-test was used with the following significance levels. The results of the Sidak's post-hoc tests were indicated as follows: p < 0.05 (*), p < 0.001 (***).
The anti-proliferative effect of CAP treatment on cells could possibly be influenced by components of the cell culture medium. In particular, chemical modifications of these components-e.g., amino acids-could influence cellular metabolism. To investigate this indirect influence, untreated HDMEC were incubated with CAP-and argon-treated cell culture media for 120 h. The number of viable cells was examined 4,24,48,72,96, and 120 h after treatment. For both CAP devices, no statistically significant difference in growth between the indirectly treated HDMEC compared to control cells was found ( Figure 1C,D).
After inhibition of HDMEC proliferation after CAP-treatment had been demonstrated, the influence of CAP on cellular metabolism was examined using MTT assay. This is an established assay to assess the general metabolic activity of viable cells. HDMEC were treated with CAP or argon gas and analyzed by MTT assay after 4, 24, 48, and 72 h of incubation. For the first two measurement points, a statistically significant reduction in cell metabolism was detected after treatment with both CAP devices ( Figure 2). This reduction was more pronounced after CAP treatment with the kINPen than after treatment with the Mini Jet. After 4 h, metabolic activities in CAP-treated HDMEC were reduced to 72.7% ± 9.0% (p = 0.041) (kINPen) and 80.2% ± 7.4% (p < 0.001) (Mini Jet) compared to the control. After 24 h, reductions to 70.2% ± 8.2% (p = 0.002) (kINPen) and 89.7% ± 6.1% (p = 0.040) (Mini Jet) were obtained. The inhibitory effect of CAP treatment was not statistically significant after longer incubation periods of 48 and 72 h. After inhibition of HDMEC proliferation after CAP-treatment had been demonstrated, the influence of CAP on cellular metabolism was examined using MTT assay. This is an established assay to assess the general metabolic activity of viable cells. HDMEC were treated with CAP or argon gas and analyzed by MTT assay after 4, 24, 48, and 72 h of incubation. For the first two measurement points, a statistically significant reduction in cell metabolism was detected after treatment with both CAP devices ( Figure 2). This reduction was more pronounced after CAP treatment with the kINPen than after treatment with the Mini Jet. After 4 h, metabolic activities in CAP-treated HDMEC were reduced to 72.7% ± 9.0% (p = 0.041) (kINPen) and 80.2% ± 7.4% (p < 0.001) (Mini Jet) compared to the control. After 24 h, reductions to 70.2% ± 8.2% (p = 0.002) (kINPen) and 89.7% ± 6.1% (p = 0.040) (Mini Jet) were obtained. The inhibitory effect of CAP treatment was not statistically significant after longer incubation periods of 48 and 72 h. The cell glucose uptake after CAP treatment was measured to further investigate the effects of CAP treatment on cell metabolism. For this purpose, the glucose concentration in the culture medium was determined 4, 24, 48 and 72 h after the treatment. The measured concentration was normalized to the number of living cells. The glucose concentration of the cell culture medium per cell differed significantly (F (1, 10) = 9.897) between CAP and the control (  The cell glucose uptake after CAP treatment was measured to further investigate the effects of CAP treatment on cell metabolism. For this purpose, the glucose concentration in the culture medium was determined 4, 24, 48 and 72 h after the treatment. The measured concentration was normalized to the number of living cells. The glucose concentration of the cell culture medium per cell differed significantly (F (1, 10) = 9.897) between CAP and the control (  To verify whether the anti-proliferative effect of CAP treatment was based on the induction of apoptotic processes, caspase-3/caspase-7 activity assays (caspase-3/7 assay) were performed. HDMEC were treated with CAP or carrier gas argon as a control and incubated for 24 h, 48 h, and 72 h. At each time, CAP-treated HDMEC demonstrated a significantly increased caspase-3 and caspase-7 signal, with lower apoptosis induction after Mini Jet treatment than after kINPen treatment ( Figure  4A,B). The activity of both caspases increased with prolonged incubation time. After 72 h, the signal strengths of activated caspase-3 and caspase-7 in CAP-treated HDMEC were 2.5 fold (p < 0.001) (kINPen) and 2.3 fold (p < 0.001) (Mini Jet) compared to control.
TUNEL assays confirmed the findings obtained by the caspase-3/7 assays. HDMEC were treated with CAP or argon gas and incubated for 24, 48, and 72 h. For treatment with both CAP devices, an increased signal of DNA fragmentation was observed ( Figure 4C,D). The TUNEL signals increased 1.4 fold (p = 0.006) (kINPen) and 1.4 fold (p < 0.001) (Mini Jet) 72 h after CAP-treatment. To verify whether the anti-proliferative effect of CAP treatment was based on the induction of apoptotic processes, caspase-3/caspase-7 activity assays (caspase-3/7 assay) were performed. HDMEC were treated with CAP or carrier gas argon as a control and incubated for 24 h, 48 h, and 72 h. At each time, CAP-treated HDMEC demonstrated a significantly increased caspase-3 and caspase-7 signal, with lower apoptosis induction after Mini Jet treatment than after kINPen treatment ( Figure 4A,B). The activity of both caspases increased with prolonged incubation time. After 72 h, the signal strengths of activated caspase-3 and caspase-7 in CAP-treated HDMEC were 2.5 fold (p < 0.001) (kINPen) and 2.3 fold (p < 0.001) (Mini Jet) compared to control.
TUNEL assays confirmed the findings obtained by the caspase-3/7 assays. HDMEC were treated with CAP or argon gas and incubated for 24, 48, and 72 h. For treatment with both CAP devices, an increased signal of DNA fragmentation was observed ( Figure 4C,D). The TUNEL signals increased 1.4 fold (p = 0.006) (kINPen) and 1.4 fold (p < 0.001) (Mini Jet) 72 h after CAP-treatment.
The migration of endothelial cells into surrounding tissues is a key event of angiogenesis and is a directed process that can be stimulated primarily by the angiogenic factor VEGF. After it was shown that CAP-treatment inhibits the proliferation of HDMEC, the influence of CAP on the migration behavior of HDMEC was also investigated. For this purpose, a migration assay was established in which HDMEC migrated through the membrane of a FluoroBlok Transwell insert (Corning, New York, NY, USA; pore size 8 µm). The number of migrated cells was determined by fluorescence microscopy. In the presence of VEGF, the migration of HDMEC was statistically significantly increased. The number of migrated HDMEC increased 9.5-fold (p < 0.001) after an incubation of 6 h (VEGF-negative: 449 ± 157, VEGF-positive: 4250 ± 495 ( Figure 5A).
Following this, the effect of CAP treatment on the migration behavior of HDMEC was examined in this setup. For this purpose, the HDMEC were treated with CAP or argon gas (control) before the migration assay. To stimulate the migration of HDMEC, VEGF was present in both approaches. After an incubation period of 6 h, a statistically significant migration inhibition in CAP-treated HDMEC was detected ( Figure 5B). The number of migrated CAP-treated HDMEC after 6 h was 3.2-fold lower than the control cells (p < 0.001) (CAP: 1287 ± 286, Control: 4129 ± 533).
To investigate the influence of CAP on the ability of HDMEC to form tubes in Matrigel, tube formation assays was performed. The total tube length after an incubation period of 6 h was analyzed. The total tube length of CAP treated HDMEC was significantly shorter than the control treated cells (p = 0.005, CTRL: 151.1 ± 48.5, CAP: 97.9 ± 42.2) ( Figure 6). Five independent experiments were performed. The results were normalized to control cells and are presented as mean ± SD. All experiments were statistically evaluated with two-way RM ANOVA with Sidak's post hoc tests before normalization. The following significance levels were defined: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).
The migration of endothelial cells into surrounding tissues is a key event of angiogenesis and is a directed process that can be stimulated primarily by the angiogenic factor VEGF. After it was shown that CAP-treatment inhibits the proliferation of HDMEC, the influence of CAP on the migration behavior of HDMEC was also investigated. For this purpose, a migration assay was established in which HDMEC migrated through the membrane of a FluoroBlok Transwell insert (Corning, New York, USA; pore size 8 µ m). The number of migrated cells was determined by fluorescence microscopy. In the presence of VEGF, the migration of HDMEC was statistically significantly increased. The number of migrated HDMEC increased 9.5-fold (p < 0.001) after an incubation of 6 h (VEGF-negative: 449 ± 157, VEGF-positive: 4250 ± 495 ( Figure 5A).
Following this, the effect of CAP treatment on the migration behavior of HDMEC was examined in this setup. For this purpose, the HDMEC were treated with CAP or argon gas (control) before the migration assay. To stimulate the migration of HDMEC, VEGF was present in both approaches. After an incubation period of 6 h, a statistically significant migration inhibition in CAP-treated HDMEC was detected ( Figure 5B). The number of migrated CAP-treated HDMEC after 6 h was 3.2-fold lower than the control cells (p < 0.001) (CAP: 1287 ± 286, Control: 4129 ± 533). To investigate the influence of CAP on the ability of HDMEC to form tubes in Matrigel, tube formation assays was performed. The total tube length after an incubation period of 6 h was analyzed. The total tube length of CAP treated HDMEC was significantly shorter than the control treated cells (p = 0.005, CTRL: 151.1 ± 48.5, CAP: 97.9 ± 42.2) (Figure 6).  . CAP inhibits the tube formation of HDMEC. HDMEC were treated with CAP or carrier gas Argon and seeded in µ -slides pre-coated with Matrigel/Medium. After an incubation period of 6 h, pictures were captured and the total tube length were analyzed with imageJ. The total tube length of the control treated HDMEC was significantly longer than the CAP treated cells (A). Six independent experiments were performed. Data are given as means ± SD. The data were statistically evaluated with the paired t-test: p < 0.01 (**). Figure  To examine a possible mutagenic effect of the CAP treatment, a hypoxanthine-guaninephosphoribosyl-transferase (HPRT) assay was carried out [39]. For this purpose, CCL-93 cells were treated with CAP or the carrier gas. Growth kinetics were carried out over 72 h. This showed a significant reduction in the number of cells after CAP treatment after 48 (p < 0.001) and 72 h (p < 0.001). After, treatment cells were incubated in medium containing 6-thioguanine (TG). TG should inhibit cell growth in non-mutated cells (Figure 7). To examine a possible mutagenic effect of the CAP treatment, a hypoxanthine-guaninephosphoribosyl-transferase (HPRT) assay was carried out [39]. For this purpose, CCL-93 cells were treated with CAP or the carrier gas. Growth kinetics were carried out over 72 h. This showed a significant reduction in the number of cells after CAP treatment after 48 (p < 0.001) and 72 h (p < 0.001). After, treatment cells were incubated in medium containing 6-thioguanine (TG). TG should inhibit cell growth in non-mutated cells (Figure 7).

Discussion
A high degree of vascularization is required to satisfy the high oxygen and nutrient requirement of tumor cells and is the most critical prerequisite for rapid and aggressive tumor growth [26]. Therefore, tumor-associated angiogenesis is a major factor in the initiation and progression of malignant tumors [25]. Tumor cells can induce angiogenesis by secreting cytokines, such as VEGF, thereby stimulating endothelial cells to proliferate and migrate [25,26,40,41]. This confirms the cell culture model established in the present study and demonstrated that the presence of VEGF significantly increased the invasiveness of HDMEC.
In addition to radiation, chemotherapy and surgical debridement, the treatment of malignant tumors can be complemented by the inhibition of tumor-induced angiogenesis [26,42]. Several drugs are currently available to suppress angiogenesis, such as the VEGF-specific antibody, bevacizumab. However, bevacizumab therapy can cause severe systemic side effects or development of resistance, which may restrict its application [43][44][45][46]. Against this background, CAP treatment can be considered a new alternative in anti-oncological therapy. It has been shown that CAP treatment can inhibit both the proliferation and the cell mobility of in vitro propagated tumor cells [35,[47][48][49].
CAP treatment of HDMEC inhibited their migration, depending on the duration of treatment and the CAP device. The inhibitory effects of the CAP treatment also have a direct influence on the tube formations of HDMEC. This suggests that CAP treatment can inhibit not only the tumor cells directly but also tumor-associated cells, such as endothelial cells. One such example is the inhibitory effect of CAP on fibroblasts [50,51]. These effects were also shown in the current study. In contrast to systemic therapies, CAP only has a locally limited effect. Other studies also indicate that CAP treatment has fewer systemic side effects than conventional therapies [52][53][54]. Another favorable property of CAP is that no resistance to CAP treatment has been observed in previous studies [52,55]. Over the entire study, no reduced CAP efficacy could be detected in the present study. Indirectly mediated anti-proliferative CAP effects have not been demonstrated. A possible explanation can be explained as the inclusion of ascorbic acid in the cell medium. This is known to reduce reactive oxygen species (ROS) and thus prevents apoptosis [56] and thus can weaken the CAP effects [51]. In addition to the migration-inhibiting effect of CAP on endothelial cells, an anti-proliferative effect on human microvascular endothelial cells was also demonstrated for the first time for both CAP devices.

Discussion
A high degree of vascularization is required to satisfy the high oxygen and nutrient requirement of tumor cells and is the most critical prerequisite for rapid and aggressive tumor growth [26]. Therefore, tumor-associated angiogenesis is a major factor in the initiation and progression of malignant tumors [25]. Tumor cells can induce angiogenesis by secreting cytokines, such as VEGF, thereby stimulating endothelial cells to proliferate and migrate [25,26,40,41]. This confirms the cell culture model established in the present study and demonstrated that the presence of VEGF significantly increased the invasiveness of HDMEC.
In addition to radiation, chemotherapy and surgical debridement, the treatment of malignant tumors can be complemented by the inhibition of tumor-induced angiogenesis [26,42]. Several drugs are currently available to suppress angiogenesis, such as the VEGF-specific antibody, bevacizumab. However, bevacizumab therapy can cause severe systemic side effects or development of resistance, which may restrict its application [43][44][45][46]. Against this background, CAP treatment can be considered a new alternative in anti-oncological therapy. It has been shown that CAP treatment can inhibit both the proliferation and the cell mobility of in vitro propagated tumor cells [35,[47][48][49].
CAP treatment of HDMEC inhibited their migration, depending on the duration of treatment and the CAP device. The inhibitory effects of the CAP treatment also have a direct influence on the tube formations of HDMEC. This suggests that CAP treatment can inhibit not only the tumor cells directly but also tumor-associated cells, such as endothelial cells. One such example is the inhibitory effect of CAP on fibroblasts [50,51]. These effects were also shown in the current study. In contrast to systemic therapies, CAP only has a locally limited effect. Other studies also indicate that CAP treatment has fewer systemic side effects than conventional therapies [52][53][54]. Another favorable property of CAP is that no resistance to CAP treatment has been observed in previous studies [52,55]. Over the entire study, no reduced CAP efficacy could be detected in the present study. Indirectly mediated anti-proliferative CAP effects have not been demonstrated. A possible explanation can be explained as the inclusion of ascorbic acid in the cell medium. This is known to reduce reactive oxygen species (ROS) and thus prevents apoptosis [56] and thus can weaken the CAP effects [51]. In addition to the migration-inhibiting effect of CAP on endothelial cells, an anti-proliferative effect on human microvascular endothelial cells was also demonstrated for the first time for both CAP devices. The selection of the appropriate treatment time is crucial, since short treatment times have hardly any anti-proliferative effects, but a sharply increased CAP treatment can damage adjacent tissue [57]. Similar to the use of pharmacological compounds, a dose-response relationship should, therefore, be defined for CAP treatment. It is known that the anti-proliferative effect of both CAP sources depends on the treatment time [34]. However, device-specific parameters are also conceivable with which a dose-response relationship may be defined, such as the voltage between the electrodes and the flow rate of the carrier gas [58]. These findings help to understand why different treatment times with both CAP devices were required to achieve an almost identical anti-proliferative effect. With the Mini Jet, the gas flow rate (1.5 L/min) was only half as high as with the kINPen (3 L/min), while the treatment time required to achieve a comparable effect was twice as long. The carrier gas and the CAP device itself represent other critical parameters on which the composition of the cold atmospheric plasma depends [55]. CAP includes charged particles and radicals as well as electromagnetic radiation [59,60]. Due to this heterogeneity of CAP composition, treatment parameters for each CAP source must be defined individually for each cell type and in clinical practice for each tumor type [34].
The anti-proliferative effect of CAP in tumor cells is mainly due to the increase in intracellular concentrations of ROS and nitrogen species (RNS) with corresponding cell biological consequences [61]. The glucose assay showed inhibition of the glucose metabolism of the HDMEC after CAP treatment, since the consumption of glucose by the CAP-treated cells was significantly suppressed up to 72 h after the treatment. Additionally, the MTT assay demonstrated that the metabolic activity [62,63] of endothelial cells was reduced up to 24 h after CAP treatment. However, the metabolism of HDMEC does not appear to be permanently restricted, as the surviving HDMEC were able to regenerate and showed a metabolic activity comparable to that of the control group after 48 h. In other endothelial cells (HUVEC), the restoration of physiological cell metabolism after CAP treatment has already been shown [64]. Comparable metabolic CAP effects have also been described in squamous epithelium cells and fibroblasts [65,66]. This demonstrated regeneration of cell metabolism could not be observed in the tumor cells examined [58,[66][67][68]. The different effects of CAP on the metabolic activity of different cell types may be explained by cell type-specific redox-protective mechanisms. In contrast to non-malignant cells, tumor cells are believed to be highly susceptible to oxidative stress [58,65,66]. It is known that oxidative stress triggers apoptosis in epithelial cells [69]. Although CAP-induced apoptosis has been demonstrated in various tumor entities [34,35,70,71], the effect in endothelial cells is still largely unexplored. Analysis of apoptotic processes revealed both increased DNA fragmentation and increased activity of caspase 3 and caspase 7 in HDMEC directly treated with CAP. Indirectly mediated anti-proliferative CAP effects were not observed in HDMEC.
Discussing the use of CAP for medical treatment, the question of the safety of this procedure always arises. This is particularly relevant for a possible intraoperative use of CAP, since non-malignant areas of the surrounding tissue can also be affected by the CAP treatment.
The reports by other authors about the impact of CAP on normal, non-malignant cells show no undesirable effects to date [72]. The control tests of this study with a non-malignant cell line (CCL-93) also showed that the CAP treatment does not have any mutagenic effects on normal cells [73].

Proliferation Assay after CAP Exposure
Cell growth was determined after 4, 24, 48, 72, 96, and 120 h using a CASY cell counter and analyzer model TT (Roche Applied Science, Mannheim, Germany) with a 150 µm capillary. For this purpose, 1 × 10 4 endothelial cells were suspended in 200 µL culture media and treated with CAP or carrier gas argon (control group) for 10 s (kINPen) or 30 s (Mini Jet). After treatment, the cell suspension was transferred to another 24-Well cell culture plate. The treatment well was rinsed with 200 µL fresh media, which was also transferred to the culture plate. Then, 800 µL fresh media was added, and cells were incubated in a humidified atmosphere at 5% CO 2 and 37 • C. Cell count was determined by suspending the cells by trypsin/EDTA (Ethylenediaminetetraacetic acid) treatment and diluting 100 µL cell suspension in 10,000 µL CASYton (Roche Applied Science, Mannheim, Germany). The measurement was performed three times with 400 µL each of this dilution and was performed in triplicate. To discriminate between cell debris, dead cells, and living cells, gates of 5.88 µm/11.13 µm were used.

Proliferation Assay after Indirect Exposure
1 × 10 4 endothelial cells were suspended in 800 µL culture media and transferred to the wells of a 24 well plate. Then, 200 µL culture medium was treated with CAP or carrier gas argon for 15 s (kINPen) or 30 s (MiniJet) in a separate 24-well plate and added to the cell suspension. Cells counts were performed at 4, 24, 48, 72, 96, and 120 h after the indirect CAP-exposure, as described in proliferation assay after direct CAP treatment.

TUNEL-Assay
1.5 × 10 4 (24 h), 1.0 × 10 4 (48 h) and 6.5 × 10 3 (72 h) cells were treated with CAP or argon for 15 s (kINPen) and 30 s (Mini Jet). Additionally, negative controls lacking fluorescent labeling and a nuclease-treated 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).

Migration-Assay
FluoroBlok Transwell inserts (Corning, New York, NY, USA; pore size 8 µm) were inserted into the wells of a 24-well cell culture plate. For the assay, 5.0 × 10 4 cells were suspended in 200 µL culture media. The wells of the cell culture plate were filled with medium with or without VEGF (26.6 ng/mL). After 6 h of incubation (37 • C, 5% CO 2 ), the insets were removed, washed twice with DPBS, and fixated with 100% Methanol. Cells were stained with DAPI (1 µg/mL) over 15 min at room temperature. The bottom membrane was cut out and transferred to microscope slides. The cover slips were mounted with 33% Glycerol. Edges were sealed with transparent nail varnish. With the BZ-II Analyzer software, the number of cells was determined.
To investigate the influence of CAP on migration, cell suspensions were treated with CAP or argon carrier gas released by the kINPen over 15 s before seeding. The subsequent assay procedure remained unchanged. The assay setup is shown in Figure 8.

TUNEL-Assay
1.5 × 10 4 (24 h), 1.0 × 10 4 (48 h) and 6.5 × 10 3 (72 h) cells were treated with CAP or argon for 15 s (kINPen) and 30 s (Mini Jet). Additionally, negative controls lacking fluorescent labeling and a nuclease-treated 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).

Migration-Assay
FluoroBlok Transwell inserts (Corning, New York, NY, USA; pore size 8 µ m) were inserted into the wells of a 24-well cell culture plate. For the assay, 5.0 × 10 4 cells were suspended in 200 μL culture media. The wells of the cell culture plate were filled with medium with or without VEGF (26.6 ng/mL). After 6 h of incubation (37 °C, 5% CO2), the insets were removed, washed twice with DPBS, and fixated with 100% Methanol. Cells were stained with DAPI (1 µg/mL) over 15 min at room temperature. The bottom membrane was cut out and transferred to microscope slides. The cover slips were mounted with 33% Glycerol. Edges were sealed with transparent nail varnish. With the BZ-II Analyzer software, the number of cells was determined.
To investigate the influence of CAP on migration, cell suspensions were treated with CAP or argon carrier gas released by the kINPen over 15 s before seeding. The subsequent assay procedure remained unchanged. The assay setup is shown in Figure 8.

4.10.
Hypoxanthine-Guanine-Phosphoribosyl-Transferase (HPRT) Assay 5 × 10 4 CCL-93 cells were suspended in 200 µL media and treated with 10 s CAP or carrier gas argon. The cells were propagated in medium containing 6-thioguanin (10 µg/mL). As control CAP and carrier gas treated cells were also propagated in medium without 6-thioguanin 24, 48 and 72 h after CAP exposure, the number of viable cells were measured with CASY cell counter and analyzer, as described under point 4.2.

Conclusions
This study demonstrated the anti-proliferative effects of CAP on endothelial HDMEC, which depend on the duration of treatment and are due to the induction of apoptosis by CAP. In addition, it was shown that the migration of endothelial cells could be inhibited directly by CAP. The results of this work show that CAP is used to suppress angiogenesis and that tumor growth and the metastatic spread of malignant tumors can be directly influenced.