The HT-1080 (ATCC CCL-121, fibrosarcoma, RD (ATCC CCL-136, embryonal rhabdomyosarcoma), and GCT (ATCC TIB-233, undifferentiated pleomorphic sarcoma/giant cell tumor) cell lines were obtained from the American Type Culture Collection. HT-1080 cells were isolated from a 35-year-old man who did not receive treatment. The cells are TP53 wild type (16). RD cells were derived from biopsy specimens of a 7-year-old woman with pelvic RMS previously treated with cyclophosphamide and radiation. GCT cells were derived from the lung of a 29-year-old man. The TP53 gene was mutated in RD (homozygous (17) and GCT (two heterozygous) cells. All cell lines were grown in medium supplemented with 10% (v/v) fetal bovine serum and penicillin–streptomycin (100 U/ml). The HT-1080 and RD cell lines were grown in Dulbecco’s modified Eagle’s medium (Thermo Fisher scientific, Waltham, USA), which was supplemented with 1% sodium pyruvate (Sigma-Aldrich, St. Louis, USA) for HT-1080 cells. GCT cells were grown in McCoy’s (Thermo Fisher Scientific, Waltham, USA). Cells were maintained at 37°C and 5% CO₂ in a humidified incubator.

Photon irradiation hereafter referred to as X was performed using an ISOVOLT 320 X-ray machine (Seifert–Pantak, East Haven, CT) at 320 kV, 10 mA with a 1.65-mm aluminum filter, and a distance around 50 cm to the object being irradiated (18).

Proton irradiation hereafter referred to as H was performed with an IBA Proteus PLUS proton therapy system (IBA PT, Louvain-la-Neuve, Belgium) at the West German Proton Therapy Centre Essen (WPE). A clinical pencil beam scanning line with an IBA PBS-dedicated nozzle was used. Several proton beams were energy and intensity modulated layered to form a spread-out Bragg peak (SOBP) consisting of five energy layers of 118.8 MeV up to 129.9 MeV. The proton beam range was compensated with a range shifting block water equivalent thickness (WET) = 74 mm, material: polymethyl methacrylate (PMMA)) and an additional solid water phantom (RW3 plates, type SP34 IBA Dosimetry, composition: 98% polystyrene + 2% TiO₂) with a WET of 3.3 cm to irradiate the cells in the middle of the SOBP. Cells in multiwell plates were irradiated with a homogeneous field with absorbed physical doses of 1, 2, 4, 6, or 8 Gy (field sizes: 20 × 20 × 1 cm³). Multiwell plates were positioned laterally and centered with the sample surface in the isocenter on the treatment table and irradiated with a gantry angle of 0°.

The cytotoxic antibiotic doxorubicin (Dox) (2 mg/ml, Medac GmbH, Wedel, Germany) was purchased from and prepared by the pharmacy of the University Hospital Essen. For experiments, Dox was diluted in PBS (Invitrogen, Carlsbad, USA) and culture medium. Cells were treated in different sequences: 3 h before irradiation (DoxA), 3 h before irradiation and refreshed within 1 h after irradiation till the end of the experiment (DoxB), or 1 h after irradiation till the end of the experiment (DoxC) (Figure 1D).

RD or GCT cells were cultured in normal growth media until confluence. The medium was collected, centrifuged, sterile-filtered (0.2 µm, Roth, Karlsruhe, Germany), and stored at −20°C until use. The conditioned medium was mixed with fresh medium as a 20% mixture for RD cells and a 40% mixture for GCT cells for the colony formation assay (19).

For the clonogenic survival, HT-1080 and GCT cells were preseeded 8 h and RD cells 24 h prior to radiation in triplicates in six-well plates. Cells were treated with Dox-containing culture medium. Following the irradiation, the media of all samples were changed with medium (HT-1080), conditioned medium (GCT and RD), or Dox-containing (conditioned) medium. The colonies were fixed after 9 (HT-1080), 10 (GCT), or 12 (RD) days depending on the cell doubling time (HT-1080: 24 h, GCT: 26 h, RD: 48 h), stained using 0.3% crystal violet dye (Roth, Karlsruhe, Germany) in 70% ethanol for 10 min at RT, rinsed with water, and air dried. Colonies with 50 cells were scored as surviving.

Cells were plated 24 h before treatment in six-well plates. Propidium iodide (PI) staining and flow cytometry analysis for apoptotic DNA fragmentation (subG1 population) were performed 48, 72, or 96 h post treatment. Cells were incubated for 15–30 min at RT with a staining solution (0.1 M Tris, 0.1 M NaCl, 5 mM MgCl₂, 0.05%, Triton X-100 (all Roth, Karlsruhe, Germany)), additional 62 µg/ml RNase A (AppliChem, Darmstadt, Germany), and 40 µg/ml PI (Sigma-Aldrich, St. Louis, USA) (20). Samples were analyzed by flow cytometry (FACSCalibur, Becton Dickinson, Heidelberg, Germany; FL-2) as described elsewhere (18). Cell-cycle phase distribution was analyzed with Kaluza software to identify the subG1 population (apoptotic DNA fragmentation, whole population), and in a second step, the living cell population (G1, S, G2/M phase) was investigated for a G2 arrest. Statistical analysis was performed in GraphPad Prism Version 8.3.0.

The migratory potential of cells was investigated with the migration assay 48 h post treatment at 0, 3, 6, 9, 24, and 48 h time points after scratch induction (Supplementary Figure 1). Wound closure was documented in images and determined by measuring the area of the scratch using ImageJ (Wayne Rasband, National Institutes of Health, US states) with the plugin Wound_healing_size_tool_updated (19). To calculate the maximum motility speed for each cell line, we calculated a simple linear regression between two time points (HT-1080: 0–3 h, GCT: 3–6 h, RD: 6–9 h) and determined the slope in the steep part of the curve. Additional morphological changes were evaluated by a sarcoma specialist on the basis of images of the migration assay.

The cell proliferation reagent WST-1 (in PBS 1:3, Roche, Rotkreuz, Schweiz) was used as a colorimetric assay for the quantification of cellular viability and cytotoxicity according to the manufacturer’s instruction (Roche, Rotkreuz, Schweiz). Optical densities were measured at 450 nm 60–90 min after incubation (BioTek Synergy H1 microplate reader, Agilent Technologies, Santa Clara, USA). Afterward, cells were fixed with glutaraldehyde (1% in PBS, Roth, Karlsruhe, Germany) for 15 min, stained with 0.5% crystal violet (CV) dye (Roth, Karlsruhe, Germany) in deionized water for 25 min, gently rinsed in water, and air dried overnight. The crystal violet dye was resolved in ECOSURF (0.2% in PBS, Roth, Karlsruhe, Germany) on a shaker for 20 min before optical density was measured at 540 nm (19). WST-CV data were normalized to 0 Gy or 0 nM controls.

Cell survival and dose response data were fitted using the linear quadratic equation:

where SF denotes the surviving fraction of cells at dose D with curve fitting parameters α and β. Non-linear regression analysis was performed on survival curves using GraphPad Prism, version 8.3.0. RBE values for protons were calculated relative to 320 kV X-rays according to

where RBE SF is the RBE at a certain survival level (SF) and D^{X SF} and D^{H SF} are the X and H dose for an iso-effect, respectively.

Statistical analyses were performed with GraphPad Prism 8.3.0, and all data points represent at least three replicates with error bars representing the standard deviation (SD). All presented data were normalized to the experiment, time, and treatment matching controls. The SD for the controls of each assay was calculated as followed. For the colony formation assay (CFA), the plating efficiency (PE) was calculated. The corresponding SD represents the relative mean of the PEs. For subG1 levels (apoptosis) and the cell-cycle phase, the SD was calculated from the mean of relative subG1 or cell-cycle phase levels. For the cell viability and proliferation assay, measurements were normalized to 0 Gy control and the corresponding SD was calculated from the relative mean of measurements. For the migration assay, the SD was calculated from the mean of relative motility. The significant level was determined by unpaired (curve comparison) or paired t-test (data point comparison) with p values > 0.05 (not significant, ns),< 0.05 (*),< 0.01 (**), and< 0.001 (***) were considered statistically significant.

The CFA is the most reliable method to quantify clonogenic growth and survival following radiation as an important endpoint of the cellular response toward cytotoxic stimuli (21). In order to determine the radiation sensitivity of the different STS cells, CFA was performed following X or H irradiation (dose range 0–8 Gy). Plating efficiencies and survival curves were calculated from surviving colony numbers, and respective curves were fitted with the linear quadratic model (LQM) for HT-1080 and GCT cells and with a semi-log line for RD cells (Figure 1A). RD cells seem to be the most radiosensitive cell line, followed by GCT and HT-1080. Of note, no significant difference in survival curves between X- and H-irradiated STS cells could be estimated (Figure 1A). A cell line comparison of the response to X or H irradiation showed that RD, followed by GCT and HT-1080, was the most radiosensitive cells to both radiation modalities (Figure 1C). The RBEα defined as the ratio of α_H/α_X shows for HT-1080 an elevated RBE of 1.3 indicating a higher sensitivity toward H irradiation (Figure 1B). This effect was not seen for GCT or RD cells. The RBE decreases with lower survival levels, which points toward a higher effectiveness for higher single X doses (≥6 Gy) relative to H irradiation in HT-1080 and GCT cells. Dox treatment at the indicated concentration (0–10 nM) alone was then used to determine respective chemosensitivities (Figure 1E). HT-1080 cells were most chemosensitive STS cells, and the maximum Dox concentration had to be reduced from 10 to 7.5 nM to archive surviving colonies. The longest Dox treatment, DoxB, reduced most effectively the cell survival in a dose-dependent manner (Figure 1D). In combination with X or H irradiation, the survival of all cell lines was even further reduced, again, with DoxB being most effective (Figures 1F, G). Of note, Dox treatment (only) before radiation (DoxA) was less effective than Dox after irradiation (DoxC) independent of cell model or radiation quality (Figure 1E). When comparing Dox treatment alone with combined treatment modalities, significant differences for HT-1080 (2 nM DoxB and X; 2 nM DoxB/C and H; 5 nM DoxC and X) and RD cells (2 nM DoxB/C and H) were revealed; GCT cells were not significantly affected. When comparing matching DoxA and DoxB or DoxC (alone or in combination with irradiation), significant differences for all Dox concentrations in HT-1080 and RD cells, for 5 nM and in GCT for 10 nM (Figures 1E–G) were evaluated.

Apoptosis is a further mechanism of cell death following radiation exposure and the main mechanism of action for the DNA damaging drug Dox (22). According to the clonogenic survival measurements performed above, apoptosis induction was analyzed next within the first 96 h following X or H irradiation and 10 nM Dox treatment by determining apoptotic DNA fragmentation using flow cytometry analysis in combination with PI staining. Relative to controls (0 Gy, 0 nM Dox), the subG1 population increased with radiation dose and time after treatment in HT-1080 and RD cells whereas in GCT cells only a radiation dose-dependent effect was seen (Figure 2A). Dox treatment alone had minor effects in HT-1080 and RD cells and did not affect GCT cells (Figure 2B). Combined X or H irradiation with Dox showed a radiation dose-dependent higher apoptosis rate and a Dox schedule-dependent difference with DoxB and DoxC being more effective than DoxA (Figures 2C–F). Matching X and H samples were compared by identifying the potential influence of the radiation quality (Supplementary Figure 2). Only the apoptosis rates in HT-1080 were statistically different following 8-Gy radiation alone (Supplementary Figure 2A). However, GCT and RD cells are shown in combination with DoxA and RD cells also in combination with DoxB significant differences following 8 Gy (Supplementary Figures 2B, C). The cellular response following DoxC was radiation quality independent (Supplementary Figure 2D). The data were normalized to the respective dose (4 or 8 Gy), radiation quality (X or H), and time matching (48, 72, 96 h) of samples to identify potential additive or synergistic effects in combined treated samples (Supplementary Figure 3). For HT-1080 cells, an additive effect could be identified for both irradiation qualities but only for DoxB and DoxC. In contrast, in GCT cells, no additional effect was seen for H irradiation and any Dox treatment. RD cells showed additive effects for X and H irradiation with DoxC (Supplementary Figure 3).

DNA damaging treatment such as radiation and chemotherapy can induce a transient or permanent cell-cycle arrest stopping the proliferation of damaged cells and providing an opportunity for repair (23). Therefore, the effect of mono- or combined treatment with Dox and X or H radiation on cell cycle phases was analyzed (Figure 3). The HT-1080 cell did not show a cell-cycle alteration within 96 h after the indicated treatments. In contrast, GCT and RD cells accumulated in the G2/M phase 48 h after treatment with radiation only, or in combination with DoxA and DoxB. For the most intense treatment (DoxB 8 Gy X or H), 37.7% and 40.2% for GCT and 48.6% and 47.3% for RD cells accumulated in the G2/M phase at 48 h, respectively. Arrests were beginning to resolve at 96 h post treatment; significant changes relative to controls could still be detected (Figure 3).

Due to the G2/M phase arrest in two cell lines (GCT and RD cells), we hypothesized that radiation might also reduce the general proliferation activity. Cellular proliferation levels following irradiation and Dox treatment were then estimated for the different STS cells using the crystal violet assay (Figure 4). Relative to controls (0 Gy, 0 nM Dox), all cell lines showed reduced proliferation activities following both radiation qualities in a dose-dependent manner (Figure 4A). Dox treatment alone—in either treatment schedule—exhibited effects on the proliferation levels of GCT cells. DoxB and DoxC treatment schedules in contrast were able to reduce proliferations in HT1080 and RD cells (Figure 4B). When using radiation treatment in addition, all combinatory treatments significantly lowered proliferation activities of all STS cell lines investigated 96 h post onset of treatment (Figures 4C-F). Time- and dose-matching X- and H-exposed samples were additionally compared by identifying the potential influence of the radiation quality (Supplementary Figure 4). No significant changes could be found with the exemption of whole curve comparison of HT-1018 cells following 4 Gy and DoxA (Supplementary Figure 4B). To identify potential additive or synergistic effects in combined treated samples, the data were normalized to the respective dose (4 or 8 Gy), radiation quality (X or H), and time matching (48, 72, or 96 h) samples (Supplementary Figure 5). GCT cells were the most affected cell line, and additive effects were found for all Dox conditions with X irradiation (Supplementary Figures 5A, B). Following H irradiation, much fewer effects could be detected. In contrast, RD cells were the least affected cell line (Supplementary Figures 5B, C). However, DoxB seems to be the most efficient for all cell lines (Supplementary Figures 5).

Cellular viabilities were measured via metabolic activities following combined treatment of X or H irradiation with Dox using the WST-1 reagent (Figure 5). Relative to controls (0 Gy, 0 nM Dox), both radiation qualities lowered the cell viability in a dose-dependent manner. GCT recovered independent of radiation quality to the control level after 4 Gy and 96 h, whereas HT-1080 and RD did not (Figure 5A). Dox alone had only minor effects on cellular viabilities; GCT cells were not affected, whereas minor effects of DoxB (HT-1080 cells) and DoxC (HT-1080, RD cells) were seen (Figure 5B). The combination of X and all Dox treatments significantly reduced the cell viability in HT-1080 and RD cells 96 h post treatment; in GCT cells, only 8 Gy X and DoxA and DoxB was effective (Figures 5C-D). To 96 h post treatment, H and Dox significantly decreased metabolic activity in all cell lines and treatments except HT-1080 to 4 Gy DoxB and DoxC (Figures 5E-F). Time- and dose-matching X- and H-irradiated samples were assessed to identify the potential influence of the radiation quality (Supplementary Figure 6). Again, no significant difference between X- and H-irradiated samples 96 h post treatment could be found, exempt GCT to 4 Gy (96 h) or 8 Gy (whole curve). In contrast, whole curve comparisons showed significant changes for HT-1080 and RD following irradiation and DoxA or DoxB. (Supplementary Figures 6B, C). To further reveal potential additive effects in combined treated samples, data were normalized to the respective dose (4 or 8 Gy), radiation quality (X or H), and time matching (48, 72, 96 h) samples (Supplementary Figure 7). For X-exposed samples, additive effects were found for HT-1080 and RD cells whereas GCT cells were not affected (Supplementary Figures 7A, B). No additive effects were identified following H irradiation and any Dox treatment (Supplementary Figures 7C, D).

The migration assay was used to study morphological changes upon treatment (Figure 6, Supplementary Figure 1). Untreated HT-1080 cells, under the given cell culture conditions, appear small, with a spindled to round shape, with aspects of a whirling architecture. The nuclei are hyperchromatic and broadly isomorphic. Upon X and H irradiation, HT-1080 cells seem slightly enlarged and appear predominantly in spindle shape with long cytoplasmatic processes; nuclei appear to be increasingly anisomorphic. No morphological changes were seen following DoxA alone or in combination with any radiation treatment, compared with X or H irradiation alone. DoxB and DoxC, however, induced spindle-shaped cells with long cytoplasmic processes and anisonucleosis. Combined treatment with DoxB or C and both radiation qualities increased the amount of anisonucleosis and increased the frequency of cells, which lost their cytoplasmic processes and their bipolar spindled shape.

Unirradiated GCT cells show a largely homogeneous spindled morphology with long cytoplasmic processes creating intercellular connections. Following X and H irradiation, cells show cytoplasmic and nuclear enlargement; furthermore, multinucleated cells appear. Few GCT cells develop a dendritic shape with fibroblastic appearance. Monotreatment with DoxA or combined treatment with any radiation and DoxA did not alter the morphology. Cells under DoxB or DoxC treatment alone appear with extended cytoplasmatic processes in GCT cells. The morphological effects upon irradiation and chemotherapy alone were also seen following combined treatment with irradiation and DoxB or DoxC.

Untreated RD cells present as networking spindled cells with long cytoplasmic processes and broadly isomorphic nuclei. In co-localization, few single polygonal cells with larger, roundish nuclei are apparent. Upon X and H irradiation, cells and nuclei appear enlarged and increasingly anisomorphic, and multinucleated cells show up. The cytoplasm becomes granular, and some cells loose the spindled morphology. Independent of treatment schedule, Dox treatment alone had no effect on the morphology of RD cells, like combined therapy with DoxA and radiation. However, exposure to combined X or H radiation and DoxB or C treatment led to the appearance of long thin processes, fibroblast-like and dendrite-like cell shapes, and increasing anisonucleosis. Overall, irradiation effects morphology of STS cells more pronounced than Dox treatment.

Finally, the migration assay was used to study cellular motilities by measuring the surface area that cells occupy over time after treatment with X or H irradiation and 10 nM Dox (Figure 6, Supplementary Figure 1). Relative to controls (0 Gy, 0 nM Dox), motilities for HT-1080 and GCT were significantly reduced following 8 Gy irradiation. However, RD cells lowered the motility only after 4 Gy X significantly (Figure 6A). Dox treatment lowered the motility in a cell line-dependent manner with HT-1080 being the most and GCT being the least affected (Figure 6B). Combined treatment of X irradiation and Dox reduced the motility in all cell lines where the 8 Gy dose was again more effective in HT-1080 and GCT, whereas RD was more affected after 4 Gy and Dox (Figures 6C, D). Interestingly, the combined treatment of Dox and H irradiation had less effects on cellular motilities. Only HT-1080 (8 Gy only) and GCT (both doses) cells showed significant effects (Figures 6E, F). The maximum speed of cell migration was calculated to form the exponential phase of the motility curves (HT-1080 cells 0–3 h; GCT cells: 3–6 h and RD cells 6–9 h). With the exemption of 4 Gy X in GCT and RD cells, the cell motility was reduced by radiation, all Dox schedules, and combined treatments relative to controls (0 Gy, 0 nM Dox, Figure 6G). The data were additionally normalized to the respective dose (4 or 8 Gy), radiation quality (X or H) (Supplementary Figure 8). Time- and dose-matching X and H irradiated samples were assessed to identify the potential influence of the radiation quality (Supplementary Figure 9). With the exemption of 4 Gy with DoxB in HT-1080 and 8 Gy with DoxC in RD, no significant influence of the radiation quality on the motility of the cells could be measured.