Synergistic effect of cold gas plasma and experimental drug exposure exhibits skin cancer toxicity in vitro and in vivo

Graphical abstract

Highlights

• •Cold gas plasma-generated ROS have anticancer potential.
• •A 154-drug library screening was performed in 3D tumor spheroids using quantitative high-content imaging analysis.
• •Candidates combining well with ROS-mediated anticancer effects were analyzed.
• •A compound was identified with low toxicity that amplified ROS-derived tumor toxicity in vitro, in ovo, and in vivo.

Abstract

Introduction

Skin cancer is often fatal, which motivates new therapy avenues. Recent advances in cancer treatment are indicative of the importance of combination treatments in oncology. Previous studies have identified small molecule-based therapies and redox-based technologies, including photodynamic therapy or medical gas plasma, as promising candidates to target skin cancer.

Objective

We aimed to identify effective combinations of experimental small molecules with cold gas plasma for therapy in dermato-oncology.

Methods

Promising drug candidates were identified after screening an in-house 155-compound library using 3D skin cancer spheroids and high content imaging. Combination effects of selected drugs and cold gas plasma were investigated with respect to oxidative stress, invasion, and viability. Drugs that had combined well with cold gas plasma were further investigated in vascularized tumor organoids in ovo and a xenograft mouse melanoma model in vivo.

Results

The two chromone derivatives Sm837 and IS112 enhanced cold gas plasma-induced oxidative stress, including histone 2A.X phosphorylation, and further reduced proliferation and skin cancer cell viability. Combination treatments of tumor organoids grown in ovo confirmed the principal anti-cancer effect of the selected drugs. While one of the two compounds exerted severe toxicity in vivo, the other (Sm837) resulted in a significant synergistic anti-tumor toxicity at good tolerability. Principal component analysis of protein phosphorylation profiles confirmed profound combination treatment effects in contrast to the monotherapies.

Conclusion

We identified a novel compound that, combined with topical cold gas plasma-induced oxidative stress, represents a novel and promising treatment approach to target skin cancer.

Introduction

Skin cancer, including melanoma and non-melanoma skin cancer (NMSC), is the most common cancer type worldwide [1]. NMSCs, including basal and squamous cell carcinomas, are the most frequently diagnosed skin cancers. By contrast, melanoma skin cancer is the most aggressive form. Patients diagnosed at late tumor stages often show high metastatic rates, leading to an estimated five-year survival rate of only 27% [2]. Recently, much progress has been achieved in developing novel anti-cancer therapies using malignant melanoma as model tumors. For instance, precision medicine approaches applying targeted therapies and immune checkpoint blockade strategies have shown remarkable efficacy in a subset of melanoma patients and have been game-changers in terms of clinical impact. Despite these successes, many tumors develop a secondary resistance against, e.g., proto-oncogene B-Raf (BRAF) inhibitor monotherapy, which limits the effectiveness of the intervention [3]. Combination therapies with two different drugs have been tested to overcome this challenge and showed improved response rates, progression-free survival, and overall survival [4]. Even though these novel therapies have revolutionized melanoma treatment, most patients with advanced skin cancer still succumb to their disease. Thus, there remains an urgent need to develop innovative treatment strategies to target skin cancer. Moreover, combination therapies should be increasingly considered for cancer treatment due to the frequent development of secondary therapy resistance of cancers to monotherapies.

It is acknowledged that both skin cancer development and therapy are subject to redox control [5], [6]. Reactive oxygen species (ROS) play critical roles in tumor cell metabolism [7], treatment responses [8], the efficacy of anti-tumor immunity [9], and the shaping of the tumor microenvironment (TME) [10]. While it is understood that chronic ROS exposure may be one of the etiological agents of skin cancer development, it has been shown that acute high doses of ROS also serve therapeutic uses. For instance, photodynamic therapy (PDT) predominantly generates singlet delta oxygen and has been approved to treat NMSC [11]. Intriguingly, several experimental and clinical physical modalities applied topically as anti-cancer agents were reported to work, at least partially, through endogenous or exogenous ROS generation [12]. It is important to note that ROS activity is subject to hormetic effects. The concept of hormesis generally states that a given agent can exert opposite effects, depending on the concentration applied being either low or high [13]. For ROS, it is described that low doses exert oxidative eustress and initiate and maintain signaling responses, while high doses are increasingly cytotoxic within so-called oxidative distress [14].

Cold gas plasma is a relatively recent technology applied in medicine. Clinically, it is approved at low doses for treating chronic wounds in Europe [15]. Such plasmas are partially-ionized gases generating a plethora of ROS simultaneously [16] identified to be central to the mechanism of action [17]. Further components include the generation of ions, electrons and both charged or neutral reactive and molecular species. In addition, electric and magnetic fields are being generated [17], [18]. Elementary plasma processes contributing to the formation of ROS/RNS take place in the nano to microseconds range. Here, highly reactive electrons and primary species, including ions and excited atoms, pass the plasma afterglow and provoke secondary species formation by energy transfer occurring through collision followed by further ionization, dissociation and excitation [19], [20]. In recent years, the technology has been increasingly investigated in oncology. Many in vitro studies and a constantly growing number of in vivo animal studies support the potential use of cold gas plasma in cancer treatment [21], [22]. In a series of patient case reports, cold gas plasma was applied in the palliative setting to reduce the microbial load and typical fetid odor of locally advanced and ulcerating squamous cell carcinomas of the head and neck. As a result of this treatment, improved quality of life due to reduced odor and pain, a reduction in microbial load, increased apoptosis in cold gas plasma-treated tumor tissues, and superficial partial remission of the tumors in one-third of the cases were reported [23], [24], [25]. Furthermore, two clinical case series revealed an impressive efficacy of cold gas plasma (partial or total remission of > 70 % of treated lesions) for the treatment of actinic keratosis, a precancerous lesion of the skin that may evolve into invasive squamous cell carcinoma [26], [27].

Based on the recent success of small molecule-based targeted cancer therapies [28], the benefits of combination treatments [29], [30], and the highly innovative potential of cold gas plasma for cancer treatment, we set out to identify novel small molecules with anti-cancer efficacy with a particular emphasis on combination effects with cold gas plasma in vitro, in ovo, and in vivo. For this, we first screened a library of 155 small molecules for their anti-skin cancer efficacy in combination with cold gas plasma. This drug screening was performed in 3D tumor spheroids, and potential candidates were validated in 2D cell cultures before the most promising candidates were further validated and characterized in 2D cell cultures, vascularized tumor organoids in ovo, and xenograft skin cancer models in vivo. This led to the identification of a candidate substance that was found to combine promisingly with cold gas plasma anti-cancer therapy.

Experimental section

Cell culture

The human melanoma cell lines A375 (Cell Lines Service (CLS): #300110; CVCL_0132) and MaMel86a (CVCL-A221), the human squamous cell carcinoma cell line A431 (CLS: #300112; CVCL_0037), the murine squamous cell carcinoma cell line SCC7 (CVCL_V412), and non-malignant human HaCaT keratinocytes were cultured in Dulbecco's Modified Eagle Medium (DMEM; ThermoFisher, Germany) supplemented with 10 % fetal calf serum (FCS) and penicillin/streptomycin (100 units/ml penicillin, 100 µg/ml streptomycin; Sigma-Aldrich, Germany) at 37 °C in humidified air with 5 % (v/v) CO₂. For cell culture experiments, the cells were incubated in fully-supplemented DMEM for 24 h before treatments were performed in serum-free medium. Fully-supplemented Roswell Park Memorial Institute medium (RPMI; ThermoFisher, Germany) was used for screening experiments with cold gas plasma due to its reduced content of anti-oxidant molecules, such as pyruvate [31].

Cold gas plasma jet and treatment

The well-characterized [32] atmospheric pressure plasma jet kINPen (neoplas, Germany) was used. The jet was operated at 1 MHz and at a power of 1 W dissipated into the cold gas plasma using argon gas (99.9999% purity; Air Liquide, Germany). Optical emission spectroscopy (OES) was performed on the cold gas plasma jet as described before [33]. Briefly, a UV-sensitive spectrometer (AvaSpec-2048-USB2; Aventes, Germany) with a spectral resolution of 0.7 nm was used end-on the cold gas plasma jet at a distance of 50 mm from the jet nozzle. Standardized treatment distance (15 mm) and gas flow rate (2 slm) were used for reproducible treatment of cells in 96-well plates using computer-aided xyz-stages (CNC, Germany). Treatment in the invasion and migration experiments was at a gas flow rate of 1.7 slm and a distance (nozzle) to the liquid surface in the inserts of ∼ 6 mm. In addition, cells were left untreated, or exposed to the working gas alone without ionization to control for gas flow pressure effects (gas control). For the in vivo studies, the argon gas flow rate was increased to 5 slm and the operating distance from the jet to the surface of the skin of tumor-bearing mice was between 8 and 10 mm to create conductive cold gas plasma jet treatment conditions that served to maximize the exposure effects in tissues [33].

Small molecules

A compound library of 155 small molecule compounds (e.g. AG635, AG387, Sm837, and IS112), composed by the Institute of Chemistry (Rostock University, Germany), was screened. For in vitro experiments, small molecules were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM. The compounds were diluted 1:100 in cell culture medium to obtain a final concentration of 100 μM, which was used to prepare appropriate further dilutions in the medium. In case of the spheroid screening experiments, this intermediate stock was then further diluted to 10, 1.0, and 0.1 µM. For in vivo application in mice (see below), first the solubility of small molecules in different solvents applicable for use in animal studies has been compared. This comparison revealed that the solubility of small molecules is best in Cremophor EL (Sigma Aldrich, Germany) and ethanol (Carl Roth, Germany). Therefore, the small molecules were dissolved in Cremophor EL and ethanol (1:1 v/v) and the solution was further diluted by adding two volumes of 0.9 % (v/v) sodium chloride (NaCl). 6-Chloro-7-methyl-3-pentafluoropropanoylchromone (IS112, C₁₃H₆CIF₅O₃) was intraperitoneally (i.p.) injected at a dose of 30 mg/kg body weight. For this, IS112 was dissolved to a concentration of 9 mg/ml and diluted in sterile 0.9 % NaCl solution to a final concentration of 3 mg/ml. As 6-Methyl-3-(2-fluorobenzoyl)chromone (Sm837, C₁₇H₁₁FO₃) is less soluble compared to IS112, it was administred via i.p. injection at a lower final dose of 10 mg/kg body weight. For this purpose, Sm837 was dissolved to a concentration of 3 mg/ml and diluted in sterile 0.9 % NaCl solution to a final concentration of 1 mg/ml. Animals in the control groups received an equal volume of vehicle (Cremophor EL and ethanol, 1:1, diluted in sterile 0.9 % NaCl solution to a final concentration of 33 % Cremophor EL and ethanol) via i.p. injection.

Compound screening using 3D tumor spheroids

Tumor spheroid formation was achieved as described before [34]. Briefly, 5 × 10³ tumor cells were seeded in ultra-low attachment plates (Nunc, Denmark) in 100 µl of fully supplemented RPMI1640 cell culture medium, and spheroids were allowed to form for 48 h. The outer wells of each 96-well plate were filled with deionized water to avoid edge effects during the cultures. The spheroids were either left untreated or treated with vehicle (solvent DMSO; final concentration 0,1%), cold gas plasma (30 s), small molecules (at a concentration of 0.1, 1.0, or 10 µM), or a combination of cold gas plasma and solvent or small molecules. Twenty-four hours later, sytox orange (final concentration 1 µM; ThermoFisher, Germany) was added to the wells for cell death detection. The plates were screened using a high-content imaging device (Operatta CLS; PerkinElmer, Germany) in non-confocal mode and with a 10x air objective (NA = 0.3; Zeiss, Germany). Fluorescence acquisition was achieved at λ_ex 505 nm and λ_em 570 nm. After spheroid object segmentation, the mean fluorescence intensity (MFI) of the area was calculated and normalized to that of untreated spheroids in each well plate.

3D organoid models

The tumor-chorioallantoic membrane (TUM-CAM) model was used as described previously [35] to study the anti-tumor effect of IS112 and Sm837 in combination with cold gas plasma. Briefly, pathogen-free fertilized chicken eggs (Valo BioMedia) were prepared, and 1 × 10⁶ cells were seeded in a plastic ring on the CAM. Solid A431 and SCC7 tumors were exposed to cold gas plasma for 1 min on day 10, with the visible cold gas plasma plume touching the tumor surface (conductive mode) [33]. Alternatively, small molecules were injected into the tumor mass as monotherapy (20 µl containing 1 mM of the compound) or administred in combination with cold gas plasma. On day 14, tumor growth was investigated ex vivo by assessing tumor weight after careful excision. To evaluate the safety and toxicity of the small molecule Sm837, hatched eggs were punctured at day 4, and 250 µg of Sm837 in 90 µl DMSO were injected into the amnion. Eggs were candled every day to check the viability of the embryos before they were sacrificed at day 12. Dosing was in accordance with the animal experiments.

Metabolic activity, cytotoxicity, and cell viability

The metabolic activity of 2D cell cultures was determined after treatment using a resazurin-based (Alfa Aesar, Germany) 96-well compatible assay as described before [36]. Briefly, 1 × 10⁴ cells were seeded per well in 100 µl cell culture medium and incubated for 24 h to allow cells to adhere. Afterward, the medium was removed, cells were washed with phosphate-buffered saline (PBS; Sigma-Aldrich, Germany), and 100 µl serum-free medium was added. Cells were treated with cold gas plasma or small molecules alone, or a combination of both. Control cells remained untreated or were exposed to the vehicle (DMSO). After 48 h incubation, 10 µl of 1 mM resazurin was added per well, and the plates were further incubated at 37 °C. The fluorescence of the metabolized resorufin was measured at λ_ex 535 nm and λ_em 590 nm using a microplate reader (Infinite F200 pro; Tecan, Switzerland). Treated sample data were normalized to vehicle controls and plotted on a log scale to generate dose–response curves for calculating corresponding IC₂₅ and IC₅₀ values. Cell viability was determined using the WST-1 assay (Sigma Aldrich, Germany) according to the manufacturer's protocol to evaluate possible cytotoxicity under conditions of the Boyden chamber assay. To this end, cells were seeded in 48-well plates at a density of 2.5 × 10⁵ cells per well in a final volume of 500 µl serum-free DMEM. Cells were directly exposed to cold gas plasma, drug monotherapy, or a combination treatment of both. The amount of viable cells was detected after incubation for 72 h. Viability was determined after addition of 30 µl WST-1 reagent by measuring absorbance at 450 nm and of a reference wavelength at 690 nm. In addition, ROS-induced apoptosis was evaluated 24 h after cold gas plasma treatment using flow cytometry. Briefly, cells were exposed to cold gas plasma for 15 s, 30 s, 45 s, or 60 s, as described above. Twenty-four hours post-exposure, cells were stained with 1 µM 4′,6-diamidin-2-phenylindole (DAPI; BioLegend, The Netherlands) to identify terminally dead cells and 0.5 µM CellEvent Caspase 3/7 detection reagent (ThermoFisher, Germany) for labeling of apoptotic cells at 37 °C for 30 min. After washing, cells were immediately acquired using flow cytometry. Gating and quantification of mean fluorescence intensities were performed using Kaluza 2.2 analysis software (Beckman-Coulter, USA).

ROS detection

For ROS detection, 2‘,7‘-dichlorodihydrofluorescein diacetate (H₂DCF-DA; ThermoFisher, Germany) was used according to the manufacturer's protocol. Briefly, 2 × 103 cells/well were seeded in 100 µl cell culture medium in 96-well plates and incubated for 24 h under cell culture conditions. Afterward, cell culture medium was removed, cells were washed with PBS, and 100 µl of 25 µM H₂DCF-DA diluted in PBS was added per well. After incubation for 45 min, cells were washed and treated with small molecules or cold gas plasma alone, or with the combination of both. Fluorescence was measured after 5 min, 1 h, 24 h, and 48 h using a microplate reader (GloMax; Promega, Germany) at λ_ex 475 nm and λ_em 500–550 nm.

Scratch-wound assay

To quantify cell motility, cells were seeded into 48-well plates at a density of 0.75 × 10⁵ cells (A431) or 1 × 10⁵ cells (A375) and allowed to grow for 48 h (A431) or 72 h (A375) in fully-supplemented DMEM. A 10 μl plastic pipette tip was used for gently scratching the cell monolayer to create a cell-free area. Subsequently, cells were washed extensively with PBS to remove cellular debris. After PBS removal, fresh serum-free DMEM was added to the cells and microscopy brightfield images were taken before as well as 6 h, 24 h, and 48 h after argon gas or cold gas plasma treatment for the indicated times to monitor the scratch-wound closure. To quantify migration, the Zen2 software (Zeiss, Germany) was used. Microscopy images of marked regions along the wounded area were obtained using an inverted microscope.

Tumor cell invasion (modified Boyden chamber assay)

Cell invasiveness was quantified by a modified Boyden chamber assay as described before [37] using Falcon cell culture inserts (8-µm pore size; Corning, Germany). In this assay, the cells seeded into the inserts must pass a matrix gel layer by proteolytic degradation and subsequently migrate through a polyethylene terephthalate membrane with 8-µm pores to a chemoattractant (DMEM 10% [v/v] FCS) in the 24-well companion plate (lower compartment). In brief, the upper sides of the inserts were coated with 70 µl (corresponds to 22.12 µg Matrigel) of a Matrigel-PBS-solution (Matrigel-PBS-mixture; 1:25 dilution; Matrigel Basement Membrane Matrix, Prod. No. 356 234, Corning, Germany) per insert and prepared 24 h prior the stimulation of A375 or A431 cells. For stimulation, cells were placed at a density of 2.5 × 10⁵ cells in a final volume of 500 µl serum-free DMEM per insert. Cells were immediately treated with cold gas plasma or small molecules alone, or a combination of both, and were incubated for 72 h. In combination treatment, the cold gas plasma exposure of cells was carried out immediately before the drug was added to the cells. After incubation, the non-invaded cells remaining on the upper side of the inserts were removed together with the matrix gel layer, and the Falcon cell culture inserts were washed with a cotton swab. Invaded cells adhering to the lower side of the insert were quantified by using the colorimetric WST-1 assay. For visualization of cells, the lower side of the transwell inserts were fixed with ethanol and stained with crystal violet. Images were acquired using a Axiovert A1 microscope with a Axiocam 105 camera and processed using ZEN2 software (all Zeiss, Germany).

Cellular adhesion capacity

The xCELLigence RTCA S16 (ACEA Biosciences, Germany) platform was used for cell adhesion measurements. The impedance magnitude (cell index) depends on the cells' number, size, and shape. The unitless adhesion capacity is calculated from the impedances at time zero (in the absence of cells) and at the time of measurement (in the presence of cells). To determine the blank value, 100 µl complete medium was added to each well of the E-Plate VIEW 16. To start a continuous measurement with a time interval of 5 min, 100 µl of cell suspension (1 × 10⁴ cells) was added, and the plate was placed in the incubator (37 °C, 5 % CO₂). Adherent cells were exposed to cold gas plasma-treated media, small molecules, or a combination of both 24 h post seeding. Data were normalized to the last-measured value before the treatment (cell index = 1.0).

Fluorescence microscopy

Sterile glass coverslips were placed in 6-well plates, and 3 × 10⁴ cells per well were seeded in 1 mL cell culture medium and incubated for 24 h under cell culture conditions. After washing the cells with PBS, the samples were treated with small molecules or cold plasma alone, or with the combination of both, and incubated for 2 h. After washing three times with PBS, cells were fixed with 4 % paraformaldehyde (Merck, Germany) in PBS for 10 min. After washing, permeabilization with 0.1 % Triton X-100 (Merck, Germany) in PBS was carried out for 10 min at room temperature. Before 10 % FCS in PBS was added for blocking for 30 min, cells were washed again. Subsequently, a 1:100 dilution of the monoclonal antibody (mouse anti-γH2A.X directly conjugated with AlexaFluor-594; BioLegend, The Netherlands) in blocking solution was added after a washing step and incubated for 1 h at room temperature in the dark. Cells were washed five times with 0.05 % (v/v) Tween 20 in PBS and three times with PBS. Coverslips were transferred from the wells upside-down on a drop of Fluoroshield mounting medium containing DAPI (Abcam, United Kingdom) on microscope slides. Finally, samples were analyzed using fluorescence microscopy (AxioVert A1; Zeiss, Germany). Quantification of fluorescence intensity was performed using ImageJ Software. Nuclear γH2A.X fluorescence was quantified in at least 34 nuclei from three images per group.

Protein phospho-kinase array

Protein phospho-kinase array analysis of 17 human MAPK pathway proteins was done as described before [38]. Briefly, A375 cells were seeded in well plates and exposed to cold gas plasma, the small molecule Sm837, or both. Afterward, cells were incubated for 1 h prior to harvesting and lysis. Next, cell lysates were washed and utilized within the phospho-kinase array (R&D Systems, Germany; Prod Nr. AAH-MAPK-1–8) according to the manufacturer's instructions.

Ethics statement

Animal experiments were approved by the State Department for Agriculture, Food Safety, and Fishery in Mecklenburg-Western Pomerania (approval code: 7221.3–1-057/18) and were conducted in accordance with the German law for animal protection (TierSchG) and the EU Directive 2010/63/EU. Humans were not involved in the study.

In vivo xenograft models

NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice were initially purchased from Jackson Laboratory. These mice develop immunodeficiency due to the severe combined immune deficiency and a complete null allele of the IL2 receptor common gamma chain. NSG mice were kept at a 12 h light/dark cycle and an ambient temperature of 21 ± 2 °C with 60 ± 20 % relative humidity. Water and standard laboratory chow were provided ad libitum. Mice were bred under specific germ-free conditions in individually ventilated cages and with environmental enrichment. For all experiments, male NSG mice aged 8 to 20 weeks and with body weights between 20 and 30 g were used. Prior to inoculation, cells were harvested using 1 % Trypsin/EDTA and stored on ice. After cell counting, cells were suspended on ice with 1:1 cold PBS/Matrigel (high concentration growth factor-reduced; Corning, Germany). For subcutaneous (s.c.) tumor cell injection, mice were anesthetized (1.5–––2.5 % isoflurane in oxygen) and kept on a heating pad (temperature of 38 °C). Both hind flanks were shaved, and 1 × 10⁶ A375 cells were injected s.c. on the left and right flanks. Small molecule or vehicle treatment started one week after tumor cell implantation and treatments were repeated every other day until day 22. Per treatment, a total volume of 10 mL/kg of body weight was administered intra-peritoneally (i.p.). Over two weeks, the mice received a total of 8 injections. Cold gas plasma treatment started on day four post tumor cell injection and was repeated every four days until day 20. It was performed for 5 min in horizontal and vertical directions, line by line, in an area of at least 1 cm², over the entire tumor. The cold gas plasma source was used at a 45° angle. In cold gas plasma-treated groups, potential pain and fixation stress under treatment was reduced using isoflurane anesthesia (performed as mentioned above). The tumor size was measured with a caliper to study the tumor growth over time. Volume calculation was obtained using the formula length × width² × 0.52. For sacrifice, mice were anesthetized by i.p. injection of ketamine of 100 mg/kg (ketamine-hydrochloride; Pharmanovo, Germany) and xylazine of 6 mg/kg (xylazine-hydrochloride; Bayer Vital, Germany). Blood was collected by retro-orbital puncture, followed by sacrifice via cervical dislocation. Afterward, tumors were harvested for further analysis.

Histology and immunohistochemistry

Half of each tumor was fixed in Formafix 4 % (buffered, stabilized with methanol; Grimm med. Logistik, Germany) for at least one day, and then prepared for paraffin embedding. For hematoxylin and eosin (H&E) staining, tumors were ultrathin-sectioned (4 µm). After deparaffinization, staining was performed, and tissue sections were assessed for routine histology. DNA fragments of apoptotic cells were enzymatically labeled and detected using the ApopTag assay (Merck, Germany), which is based on the TUNEL method. Staining was performed as per manual, followed by counterstaining with Mayer's Hemalaun solution (Merck, Germany). All slides were embedded in X-TRA Kitt (Mediate, Germany). Cells positively staining for ApopTag were quantified at 400 × magnification (Axiskop 40; Zeiss, Germany) by evaluating 5 × 100 nuclei in each of five representative areas along the invasive vital front of the tumor and expressed as positive cells (%) per tumor (for two tumors, only 2 × 100 nuclei could be counted because the tumor area was too small).

Statistical analysis

Statistical analysis, IC₂₅/IC₅₀ calculation, and graphing were performed using Prism 9.4 (GraphPad Software, USA) and t-test or one- or two-way analysis of variances (ANOVA) as indicated in the figure legends. The required number of mice was calculated before starting the experiments by sample size calculation (alpha = 0.05, beta = 0.20, power = 0.8). Based on this calculation, mice were divided into six experimental groups of 8 individuals. Animals were allocated to the groups using randomly generated numbers (random number generator; Stat Trek, https://stattrek.com/statistics/random-number-generator.aspx, the site was accessed on May 7, 2021, at 10:31 a.m.). Data show mean ± standard deviation (SD) if not indicated otherwise in the figure legends. Levels of significance were indicated as follows: p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***).

Results

Cold gas plasma provides anti-cancer activity in vitro

Before investigating the combined effects of cold gas plasma with small molecules, the biological consequences of the former were tested in vitro using the atmospheric argon plasma jet kINPen (Fig. 1a). The cold gas plasma jet produces ROS that can be made visible using optical emission spectroscopy (Fig. 1b). At 24 h following cold gas plasma exposure in vitro, metabolic activity of several cell lines was tested using a metabolic activity assay based on resorufin fluorescence (Fig. 1c). Normalized quantification showed a treatment time-dependent metabolic activity decline in all skin cell lines investigated. The squamous cell carcinoma (SCC) cell line A431 was more robust than non-malignant HaCaT keratinocytes, compared to which, in turn, MaMel-86 and A375 melanoma cell lines were more sensitive in terms of metabolic activity reduction (Fig. 1d). In parallel, a profound inhibition of invasiveness was determined by crystal violet staining of invaded cells on the lower side of transwell inserts in a modified Boyden chamber assay (Fig. 1e) and when invaded cells were quantified via WST-1 assay (Fig. 1f). Under similar conditions, a decrease in cell viability was observed as a function of exposure duration (Fig. S1), but this less pronounced than the inhibition of invasiveness observed at the respective exposure times. This suggests that the decrease in invasion in response to cold gas plasma treatment is not only due to cytotoxic effects. The observed partial toxicity may be due to apoptosis induction as an activation of effector caspases 3 and 7 was observed (Fig. S2). Next, the consequences of cold gas plasma exposure on cellular migration and motility was investigated (Fig. 1g), and found to be reduced significantly 24 h and 48 h post-treatment (Fig. 1h). Based on these findings, a low dose (30 s) cold gas plasma exposure time in combination with increasing small molecule concentrations (0.1 µM, 1.0 µM, or 10 µM) was chosen for the screening campaign in A375 3D tumor spheroids (Fig. 1i).

Fig. 1.

Study design and cold gas plasma toxicity. (a) schematic (left) and photographic (right) images of the atmospheric pressure argon plasma jet kINPen used in this study; (b) optical emission spectra of the kINPen plasma; (c) representative photographic result of the resazurin metabolic activity assay; (d) normalized metabolic activity of three skin cancer and one non-malignant skin cell line (HaCaT) following cold gas plasma exposure (grey area represents treatment times that inhibit ≥ 75% of the cells); (e) representative crystal violet staining of invaded cells at the lower side of the insert (Boyden chamber); (f) quantification of invasion 72 h post cold gas plasma treatment (mean ± standard error of the mean (SEM)); (g) representative scratch assay microscopic images; (h) scratch assay quantification (mean ± SEM); (i) scheme of drug-screening and subsequent validation and characterization. Data represent mean values of at least triplicates of three independent experiments (d,e,f) or of one experiment (g,h) per cell line. Statistical analysis was performed using two-way analysis of variance (*p < 0.05, **p < 0.01, ***p < 0.001). Scale bars are 100 µm. Gas = carrier gas without ionization. PL = cold gas plasma. Untr. = untreated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Drug screening in 3D tumor spheroids revealed two promising compounds with potent anti-cancer activity in combination with cold gas plasma

A compound library of 155 molecules with various structures (Fig. S3a) was screened in vitro in A375 3D tumor spheroids alone (at three concentrations) or in combination with cold gas plasma treatment (also, at three drug concentrations). In addition, a DMSO vehicle control (corresponding to the amount of the highest drug concentration samples) was included alone or combined with cold gas plasma exposure. Terminally dead cells 24 h post-exposure were identified using high-content imaging and algorithm-driven object segmentation and image quantification. Some compounds showed a lack of toxicity when given alone (Fig. 2a), while others combined with cold gas plasma to potentiate toxicity (Fig. 2b). Compounds that did not show cytotoxic effects, such as AG484F and AG406 (Fig. 2c, upper row), were excluded. Compounds, such as the nitrogen containing heterocycles 1256EA and 1232EA that appeared cytotoxic (Fig. 2c, lower row), which, in fact, was due to increased autofluorescence (Fig. S3b), were excluded as well. Of those compounds revealing anti-tumor toxicity alone and in combination with cold gas plasma exposure in this 3D tumor spheroid screening, two nitrogen containing heterocycles (AG635 and AG387) and two 3-acylchromones (Sm837 and IS112) (Fig. 2d) were selected for further in vitro validation in 2D cell cultures. The strongest effect on the metabolic activity decline (inhibitory concentration 25, IC₂₅) of the melanoma cell line A375 was observed after treatment with IS112 (Fig. 2e). Treatment with AG387 revealed a cytotoxic effect starting with a concentration of 25 µM, and for Sm837 and AG635, at least 50 µM was required to achieve IC₂₅ effects. All four compounds showed at least an additive effect when combined with cold gas plasma in A375 cells (Fig. 2f). Similar results were obtained after treatment of the squamous cell carcinoma cell line A431. At IC₂₅ concentrations, IS112 and Sm837 showed the lowest increase in terminally dead cells in non-malignant HaCaT keratinocytes (Fig. S3c). Therefore, further investigations were focused on IS112 and Sm837. These two compounds administered alone did not or just slightly increase intracellular ROS production, while cold gas plasma exposure markedly elevated ROS levels (Fig. 3a). The combined drug and cold gas plasma exposure further augmented intracellular ROS to some extent. ROS, also derived from cold gas plasmas, are known to elicit antioxidant phase II pathways and the DNA damage response (DDR) [39], [40], also independent of acute DNA damage [41]. To this end, histone 2A.X phosphorylation (γH2A.X), a marker known to indicate DNA damage in radiation biology and oxidative stress [42], was assessed (Fig. 3b). Quantification revealed significantly increased intra-nuclear γH2A.X levels in response to cold gas plasma exposure. At the same time, drug addition alone was less potent in elevating H2A.X phosphorylation, matching results observed with ROS accumulation (Fig. 3c). In combination, however, all combination treatments (except IS112 and cold gas plasma exposure of A375 cells) showed further enhanced γH2A.X in nuclei. Furthermore, the basis for the development and maintenance of a functioning cellular network is the adhesion of cells to surrounding cells and to the surrounding extracellular matrix. The effect of both compounds on cell proliferation and adhesion was pronounced when combined with cold gas plasma treatment in the two cell lines investigated (Fig. 3d). While mono treatments only altered the cellular adhesion capacity of A375, combination with cold gas plasma led to significantly reduced cell adhesion in both cell lines. The tumor cells' ability to invade into tissues is also related to cell adhesion, which can be investigated using a Matrigel-modified Boyden chamber assay. The results for IS112 and Sm837 were inconclusive, as a clear dose–response relationship was not observed and because the results were divergent in each cell line investigated (Fig. 4a) at generally sub-toxic concentrations utilized (Fig. 4b). Enhancement of the anti-invasive properties of cold gas plasma treatment (10 s) by IS112 and Sm837 (both: 10 µM) was not observed in A375 and A431 cells (Fig. 4c). By contrast, notable cytotoxic effects were observed only for IS112 treatments under these conditions while viability analysis of Sm837 treatments revealed a similar effect pattern of cold gas plasma and drug combinations (Fig. 4d).

Fig. 2.

Drug screening. (a-b) representative 3D A375 tumor spheroid images with nuclei (Hoechst, blue) and dead cells (sytox orange, orange) after exposure to different compounds, AG406 (a) and Sm837 (b), and cold gas plasma; (c) selected compounds with no intrinsic toxicity (AG484F, AG406) or false-positive toxicity (1256EA, 1232EA); (d) selected compounds with either intrinsic and/or combined toxicity with cold gas plasma; (e) validation of four compounds in 2D skin cancer cells and IC₂₅ calculation; (f) combination treatment of cold gas plasma (IC₂₅) and four selected compounds. Data represent mean values of at least three independent experiments. Statistical analysis was not carried out in this experiment. Scale bars are 150 µm. PL = cold gas plasma. Untr. = untreated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3.

ROS generation, histone 2A.X phosphorylation, and cell adhesion. (a) ROS generation determined via H₂-DCF-DA at different time points following exposure to cold gas plasma and drug mono (IC₂₅) or combination treatment (mean ± SEM); (b) representative fluorescence microscopy images of nuclei (blue) and γH2A.X staining (red); (c) nuclear γH2A.X fluorescence staining quantification in A375 (left) and A431 (right) cells following exposure to cold gas plasma and drug mono (IC₂₅) or combination treatment; (d) cell adhesion indexing using impedance measurements up to 48 h post-exposure to cold gas plasma and drug mono (IC₂₅) or combination treatments performed at 24 h post-seeding (vertical line). Data represent mean values of at least three independent experiments. Arrows mark the timepoints where adhesion alterations start to be significant compared to the untreated group. Statistical analysis was performed using two-way analysis of variance (*p < 0.05, **p < 0.01, ***p < 0.001). Scale bars are 10 µm. PL = cold gas plasma. Untr. = untreated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4.

Mono and combination treatment tumor cell invasion and viability. Invasion (a; mean ± SEM) and viability (b) of drug-treated skin cancer cells in a modified Boyden chamber assay (a) or WST-1 test (b) in vitro at different concentrations 72 h post exposure; invasion (c; 10–90 % percentile box plots) and viability (d) of skin cancer cells 72 h after mono and combination treatments of cold gas plasma (10 s) and drug (10 µM each) in vitro. Data represent mean values of three independent experiments (except A431 cells treated with Sm837 in (a) (two independent experiments)) carried out in at least triplicates. Statistical analysis was performed using two-way analysis of variance (a,b) or unpaired t-test (c,d) (*p < 0.05, **p < 0.01, ***p < 0.001). Gas = carrier gas without ionization. PL = cold gas plasma. Untr. = untreated.

Superior tumor organoid and xenograft toxicity using Sm837 and cold gas plasma

After comprehensive in vitro toxicity testing of IS112 and Sm837 in combination with cold gas plasma, we first aimed to confirm the anti-tumor efficacy in ovo. In this model, solid tumors are grown on chicken embryos' chorioallantoic membrane (CAM) (Fig. 5a) to establish vascularized 3D cancer organoids (Fig. 5b), which were mono or combination reated four days post-fertilization. Comparing tumor size (Fig. 5c) and weight (Fig. 5d) revealed only modest tumor reduction following mono treatments when investigating the two SCC cell lines. By contrast, the Sm837 combination with cold gas plasma treatment provided superior anti-cancer effects in both cancer cell lines. Anti-tumor combination treatment using IS112 was improved only in SCC7 cells. Since Sm837 showed good combination therapy effects, its developmental (Fig. 5e; day 4 injection) toxicity was investigated by monitoring chicken embryo survival until day 14. It should be noted that the animals received large doses (a single 250 µg injection into the amnion), which reduced survival significantly (Fig. 5f). Nevertheless, motivated by the anti-tumor efficacy of the small molecule compounds IS112 and Sm837 in combination with cold gas plasma, we aimed to validate these findings further using an in vivo xenograft mouse model carrying A375 tumors (Fig. 6a). Interestingly, and opposite to the in ovo results, Sm837 repeatedly injected into mice (8 injections à 10 mg/kg body weight) was well tolerated (no drug-induced animal death), while IS112 (up to 8 injections à 30 mg/kg body weight) was severely toxic (Fig. 6b). Using caliper measurements of tumors on both flanks, a strong tumor decline was found for cold gas plasma, IS112 alone, and the combination treatments (Fig. 6c). However, it should be noted that none of the mice survived any treatment condition involving IS112 administration. By contrast, cold gas plasma treatment halved the tumor volume in mice receiving Sm837. This effect was synergistic when measuring the area under the curve (AUC) of tumor volume (Fig. 6d). Immunohistochemical analysis of tumor tissue apoptosis (Fig. 6e) revealed a significant increase of apoptotic cells in cold gas plasma-treated tumors compared to untreated control tumors (Fig. 6f). In turn, neither Sm837 mono nor combination therapy with cold gas plasma treatment led to similarly pronounced apoptosis, making additional mechanisms or cell death pathways likely to contribute to the combined anti-tumor activity observed with the combination treatment. To this end, we performed phospho-kinase screening arrays of cold gas plasma and Sm837 mono and combination-treated A375 cells at 2 h in vitro (Fig. 7a). Sm837 treatment resulted in (>1.25-fold) increased heat-shock protein (HSP)27 and protein kinase B (AKT) phosphorylation, similar to the combination treatment. Cold gas plasma treatment solely gave (>1.25-fold) augmented phosphorylation of mammalian target of rapamycin (mTOR) and a (>-1.25) decreased amount of phosphorylated p38-mitogen-activated protein kinases (MAPK) (Fig. 7b). Principal component analysis (PCA) revealed stronger degrees of dissimilarities between all treatments (Fig. 7c), suggesting multiple pathways to participate in the results observed for combination therapy.

Fig. 5.

Anti-tumor effect of cold gas plasma and drug mono and combination treatment in ovo. (a) study design of in ovo experimentation; (b) representative photographic (top) and schematic (bottom) image of tumor growth on the chicken chorioallantoic membrane (CAM); (c) representative tumors harvested at the end of the experiment; (d) tumor weight after mono (1 mM) and combination treatment of two skin cancer cell organoids (mean ± SEM); (e) Kaplan-Meyer plot of chicken embryos injected with Sm837 (250 µg) on day 4 post-fertilization (developmental toxicity) and probability of survival (p.o.s.); (f) survival of chicken embryos injected with Sm837 (250 µg) on day 4 post-fertilizationand assessed at day 14. Statistical analysis was performed using t-test (*p < 0.05, **p < 0.01). PL = cold gas plasma. Untr. = untreated.

Fig. 6.

Anti-tumor effect of cold gas plasma and drug mono and combination treatment in vivo. (a) study scheme; (b) Kaplan-Meyer plot (survival) of mice left untreated or exposed to cold gas plasma and drug mono (IS112: 30 mg/kg; Sm837: 10 mg/kg) or combination treatment; (c) tumor volume development throughout the experiment for IS112 (30 mg/kg) or Sm837 (10 mg/kg) mono and combination treatment, respectively (mean ± SEM); (d) area under the curve of (c); (e) representative images of tumor tissue sections stained with hematoxylin and eosin (H&E) with ApopTag-labelled apoptotic cells (black arrow-heads); (f) apoptosis quantification. Statistical analysis was performed using one-way analysis of variance (*p < 0.05, ***p < 0.001). PL = cold gas plasma. Untr. = untreated.

Fig. 7.

Phosphokinase screening of Sm837 treatment. (a) representative control (top) and combination-treated (bottom) cells and their cell lysates in phosphor-kinase screening blots; (b) quantification and normalization of spot intensities in A375 skin cancer cells (mean ± SEM); (c) principal component analysis of phospho-kinase data for mono and combination treatments. PL = cold gas plasma. Untr. = untreated.

Altogether, a high-content imaging screening campaign in 3D tumor spheroids searching for anti-cancer candidates combining with cold gas plasma exposure in vitro, in ovo, and in vivo revealed an experimental drug - a chromone derivative (Sm837) - that exhibits little cytotoxicity alone but shows a remarkable synergistic anti-tumor efficacy in cold gas plasma combination therapy.

Discussion

In this study, we have identified an experimental drug that exhibits remarkable synergistic anti-tumor efficacy in combination with cold gas plasma exposure, a ROS-generating technology with anti-skin-cancer potential [43]. The identified chromone derivative Sm837 shows relatively little toxicity when applied alone to tumor cell cultures, non-malignant HaCaT keratinocytes, vascularized 3D tumor organoids, and mice with tumor xenograft skin cancers. This limited toxicity and its strong synergistic effect in combination with cold gas plasma treatment make this compound a potential candidate for future investigation for skin cancer treatment. Hence, when using a screening approach to search for drugs to be used in combination with other treatment approaches, it could be indicative to test drug candidates in combination with the other treatment instead of assessing the drug efficacy separately with mono treatment schemes only. Screening the drugs separately for the ones with the strongest efficacy may leave out those drugs that exhibit good synergistic effects with other treatment modalities while little systemic toxicity when administered alone. Previously, combination drug screening campaigns were tested for other malignancies, too, using microscopy and high-content screening approaches [44], [45]. In addition, it is undisputed that combination therapies are the future in oncology, especially those targeting multiple pathways or mechanisms of action, such as conventional chemotherapy and targeted therapy combined with immunotherapies [46]. Similar to photosensitizers in photodynamic therapy (PDT), non-toxic compounds that unleash toxic reactions in response to a local light treatment, redox-amplified drug compounds seems an ideal promotor to potentiate cold gas plasma-mediated anti-cancer effects locally.

In general, our work also confirms the potential of cold gas plasma as an anti-cancer therapy and its ability to act together with drug treatments. In contrast to other innovative ROS-based treatment approaches, including PDT, sono- [47], chemo- [48], and radiodynamic therapy [49], gas plasmas are exceptional in generating a plethora of reactive oxygen and nitrogen species locally, at high cost-efficacy, and seemless applicability. Variation of the operating feed gas further allows tuning ROS and reactive nitrogen species (RNS) chemistries for specific applications [38], [50], [51]. Among available plasma devices, plasma jets are widely used in biomedical applications. The small surface contact area enables treatment with high flexibility which is beneficial for skin cancer therapy while minimizing side effects by limiting exposure to the disease area. Additive or synergistic efficacies of cold gas plasmas have been described before, for example, in combination with doxorubicin [52], [53], [54], cisplatin [55], [56], [57], [58], methotrexate [59], imiquimod [50], gemcitabine [60], [61], olaparib [62], ADDA5 [63], cyclophosphamide [64], paclitaxel [59], [65], [66], [67], and temozolomide [68], [69], [70], [71], [72], [73], [74], [75] across different tumor cell types in vitro and partially also in vivo. Therefore, systemic drug treatment combined with local cold gas plasma treatment may provide a viable strategy to achieve improved anti-tumor efficacies with reduced systemic side effects, provided a local tumor treatment is achievable, as in skin cancer. It is understood, however, that most patients die due to hard-to-reach metastasis and not because of topical tumor burden in the skin, which would be relatively simple to remove surgically. As recently reviewed, systemic action could be achieved by the immuno-stimulative potential cold gas plasma technology displayed in several syngeneic animal models [22]. In addition, a small cohort of patients with irresectable SCC of the head and neck or palliative patient cohorts with infected superficial tumors may benefit from combined drug and cold gas plasma therapy [76].

We identified the most promising experimental anti-cancer compounds combining with cold gas plasma through a screening approach. The two identified and further explored compounds IS112 and Sm837 were chromone derivatives with different toxicities in vitro and in vivo when used as mono-treatment. The compound IS112 showed a higher cell and animal toxicity than Sm837. In general, chromones are naturally occurring heterocyclic compounds that contribute to colors in plants and exhibit a wide range of biological activities, including anti-tumor, anti-microbial, anti-viral, anti-inflammatory, anti-oxidant, anti-allergic, and anti-diabetic activities [77]. The anti-cancer activity of natural and synthetic chromones has been attributed to various mechanisms, including cytotoxicity, anti-metastasis, anti-angiogenesis, chemoprevention, and immune regulation [78]. Chromones have also been described as privileged scaffolds in anti-cancer drug discovery [79], and a variety of targets such as protein kinases, protein tyrosine phosphatases, thymidine phosphorylase, carbonic anhydrase, nuclear factor k-light-chain-enhancer of activated B cells (NF-κB), sirtuins, topoisomerases, and A3 adenosine receptors have been investigated [80]. Patil and colleagues recently provided a literature summary of 64 natural chromones, their source, and their cytotoxic activity toward different cancer cell lines [79]. In line with our findings, this overview demonstrates the broad range of anti-cancer activities of different chromone derivatives, with family members having broad effects showing low to high cytotoxicity.

Although we performed several experiments to assess the effects of Sm837 in combination with cold gas plasma on cellular processes, the mechanism of action of the synergistic effect is still to be elucidated. We observed a temporary increase of ROS levels after combination treatment, similar to the increase observed after cold gas plasma treatment alone. Along similar lines, elevated γH2A.X levels observed in combination regimes in our study have been described after cold gas plasma treatment before [41]. Originally, γH2A.X was identified as an early event after direct formation of DNA double-strand breaks. However, low γH2A.X levels are considered indispensable for mitochondrial integrity and cell cycle signaling, and are associated with apoptosis induction [81], [82], [83]. It was recently shown that exogenous ROS lead to H2A.X phosphorylation only in case of apoptosis induction [84], indicating a role in antioxidant defense signaling [85]. Since Sm837 treatment alone did not increase ROS levels, it is conceivable that Sm837 inhibited mitochondrial ABC transporters, such as ABCB10, that play a role in the protection from oxidative stress in mammalian cells. Furthermore, chromone derivatives such as MBL-II-141 have been developed to specifically act as selective and non-toxic inhibitors of the ABC transporter ABCG2 [86], [87].

In order to gain insights into how the combination treatment inhibits tumor growth, we assessed apoptotic cells in tumors after treatment. This analysis showed fewer apoptotic cells in tumors treated with the combination of Sm837 and cold gas plasma than in tumors treated with cold gas plasma alone. This may be due to early non-apoptotic cell death, proliferation stop, cellular senescence, or inhibition of angiogenesis in tumors treated with the combination therapy. Future studies need to unravel the detailed mode of action of Sm837 in combination with cold gas plasma exposure. Despite the incremental need to optimize oncological treatment strategies in terms of patient outcomes, health care is challenged by a decreasing cost efficacy of current and novel therapies, causing economic burden to societies worldwide. Here, patient stratification and implementation of effective primary, secondary, and tertiary preventive strategies are crucial to address this topic [88], [89]. Since cold gas plasma technology is economic in operation costs, it could contribute to anti-cancer care in low-to-medium income countries. In addition, from dermatology studies, clinically approved gas plasma devices have been shown to be safe and void of severe long-term side effects [15], [90].

In summary, this study identified a promising combination of a chromone derivative and cold gas plasma exhibiting a synergistic anti-cancer efficacy in vitro, in ovo, and in vivo.

Conclusion

Starting with a screening approach of a compound library and subsequent in vitro validation of the most promising candidates, two small molecule compounds were identified for further characterization in combination with cold gas plasma treatment against skin cancer. While IS112 showed better anti-tumor efficacy in vitro and in vivo as a monotherapy, it also caused severe toxicity in mice. Sm837, however, exhibited low systemic toxicity and relatively little anti-tumor efficacy alone but strong synergistic efficacy in combination with cold gas plasma treatment. The beneficial tolerability and strong targeted anti-tumor efficacy combined with cold gas plasma make Sm837 an interesting compound for skin cancer treatment. Considering the need for novel, innovative therapies for skin cancer treatment, and accounting for the advantages of combination therapies concerning secondary therapy resistance, this study not only provides a promising drug combination with cold gas plasma, but also delineates a strategy from an initial 3D screening approach over in vitro validation to in ovo and in vivo confirmation.

Funding

This joint research project “ONKOTHER-H” is supported by the European Social Fund (ESF), reference: ESF/14-BM-A55-0001/18, ESF/14-BM-A55-0002/18, ESF/14-BM-A55-0003/18, ESF/14-BM-A55-0004/18, ESF/14-BM-A55-0005/18, ESF/14-BM-A55-0006/18 and the Ministry of Education, Science, and Culture of Mecklenburg-West Pomerania, Germany. SE and LB are further supported by the German Research Foundation (DFG, EM 68/13–1), the German Federal Ministry of Education and Research (BMBF, 16GW0345), the Ministry of Commerce, Occupation, and Health of Mecklenburg-West Pomerania, Germany (TBI-V-1–349-VBW-120), the European Fond for Regional Development (GSH-20–0054), and by the Ministry for Commerce and Energy¸ Germany (03TN0019B). SB received support from the German Federal Ministry of Education and Research (BMBF, 03Z22DN11 and 03Z22Di1) and the Head and Neck Cancer Foundation Germany (Stiftung Tumorforschung Kopf-Hals).

Compliance with Ethics requirements

Animal experiments were approved by the State Department for Agriculture, Food Safety and Fishery in Mecklenburg-Western Pomerania (approval code: 7221.3-1-057/18) and were conducted in accordance with the German law for animal protection (TierSchG) and the EU Directive 2010/63/EU. Human material was not part of this study.

CRediT authorship contribution statement

Lars Boeckmann: Methodology, Validation, Formal analysis, Data curation, Project administration, Supervision, Visualization, Writing - original draft, Writing - review & editing. Julia Berner: Software, Validation, Formal analysis, Investigation, Conceptualization, Data curation, Methodology, Visualization, Writing - original draft, Writing - review & editing. Marcel Kordt: Software, Validation, Formal analysis, Investigation, Methodology, Visualization, Writing - review & editing. Elea Lenz: Formal analysis, Investigation. Mirijam Schäfer: Formal analysis, Investigation. Marie–Luise Semmler: Formal analysis, Investigation. Anna Frey: Investigation. Sanjeev Kumar Sagwal: Formal analysis, Investigation. Henrike Rebl: Methodology, Software, Validation, Formal analysis, Supervision, Writing - original draft, Writing - review & editing. Lea Miebach: Investigation, Methodology, Software. Felix Niessner: Investigation. Marie Sawade: Formal analysis, Investigation. Martin Hein: Resources, Supervision. Robert Ramer: Methodology, Software, Validation, Formal analysis, Data curation, Investigation, Supervision, Visualization, Writing - original draft, Writing - review & editing. Eberhard Grambow: Methodology, Supervision. Christian Seebauer: Supervision. Thomas von Woedtke: Funding acquisition, Writing - review & editing. Barbara Nebe: Supervision, Funding acquisition, Project administration, Resources. Hans-Robert Metelmann: Conceptualization, Funding acquisition, Resources. Peter Langer: Funding acquisition, Resources. Burkhard Hinz: Supervision, Funding acquisition, Project administration, Resources, Writing - review & editing. Brigitte Vollmar: Conceptualization, Funding acquisition, Project administration, Resources, Supervision. Steffen Emmert: Conceptualization, Funding acquisition, Project administration, Resources, Supervision. Sander Bekeschus: Conceptualization, Methodology, Software, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, 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.

Appendix A. Supplementary material

The following are the Supplementary data to this article: