A chondrosarcoma cell line, SW1353 (CLS Cell Lines Service GmbH, Eppelheim, Germany), was cultured in RPMI-1640 medium (Roswell Park Institute Medium 1640, Sigma-Aldrich) supplemented with 10% fetal calf serum, 2 mM L-glutamine and 1% antibiotics (Penicillin-Streptomycin solution, Sigma-Aldrich). A chondrocyte cell line, T/C-28A2 (a gift from Prof. Mary B. Goldring, Hospital for Special Surgery, Weill Medical College of Cornell University, New York, NY, USA), was cultured in the same medium as SW1353 cells. All experiments were performed in a humidified atmosphere with 5% CO${}_2$ and 2% O${}_2$ at 37 °C, in a Heracell™ 150i Tri-Gas incubator.

X-ray irradiations were performed with a PxiXradSmart 225cX irradiator using a tube tension of 225 kV, as previously described [21, 22, 23, 24, 25, 26, 27, 28, 29, 30], and C-ion irradiations were performed in the GANIL facility (Caen, France) using the high-energy beam line IRABAT, as previously described [31, 32]. For C-ions, a LET of 73 keV/$\mu$m was selected to reproduce clinical conditions. This LET was obtained from a native C-ion beam of 95 MeV/A, using a PMMA (Polymethacrylate of methyl) degrader inserted between the beam exit and the sample holder. To increase dose precision, all irradiations with C-ions were performed before the Bragg peak with a single energy beam (no SOBP).

Irradiated SW1353 cells and T/C-28A2 bystander cells were plated in T25 cm${}^2$ flasks. Immediately after irradiation, the cell medium was exchanged for fresh medium, and after 24 h in contact with irradiated cells, this medium was collected (Fig. 1). The conditioned medium was then centrifuged (2000 g) and transferred to flasks of the same size (T25 cm${}^2$) containing bystander T/C-28A2 cells. Bystander cells were kept in contact with the conditioned medium for 24 h and then harvested as described in Supplementary Fig. 1. The cell pellet was washed with PBS, and the dry pellet was kept at –80 °C until protein extraction.

Proteins were extracted from bystander chondrocytes (dry pellet) in lysis buffer (RIPA made without protease, in order not to interfere with proteases mass spectrometry) and left on ice for 20 min [29]. Mechanical grinding was carried out using a hand-held motorized pellet pestle for 20 s on ice, followed by 20 min at rest. The sample was then centrifuged (14,000 rpm, 20 min), and the supernatant containing the proteins was transferred to a clean tube and stored at –80 °C. Protein quantification was performed using a Bradford kit (Thermo Scientific, Waltham, MA, USA).

Five micrograms of each protein extract was prepared using a modified gel-aided sample preparation protocol [33]. Samples were digested with trypsin/Lys-C overnight at 37 °C. For nano-LC fragmentation, protein or peptide samples were first desalted and concentrated onto a $\mu$C18 Omix (Agilent) before analysis.

The chromatography step was performed on a NanoElute (Bruker Daltonics, Bremen, Germany) ultra-high-pressure nano flow chromatography system. Approximately 200 ng of each peptide sample was concentrated onto a C18 pepmap 100 (5 mm $\times$ 300 $\mu$m i.d.) precolumn (Thermo Scientific, Waltham, MA, USA) and separated at 50 °C onto a reversed phase Reprosil column (25 cm $\times$ 75 $\mu$m i.d.) packed with 1.6 $\mu$m C18 coated porous silica beads (Ionopticks). Mobile phases consisted of (A) 0.1% formic acid, 99.9% water (v/v) and (B) 0.1% formic acid in 99.9% ACN (v/v). The nanoflow rate was set at 400 nL/min, and the gradient profile was as follows: from 2 to 15% B within 60 min, followed by an increase to 25% B within 30 min, further to 37% within 10 min, followed by a washing step at 95% B and reequilibration.

Mass spectrometry experiments were carried out on a TIMS-TOF pro mass spectrometer (Bruker Daltonics, Bremen, Germany) with a modified nano electrospray ion source (CaptiveSpray, Bruker Daltonics, Bremen, Germany). A 1600 spray voltage with a capillary temperature of 180 °C was typically employed for ionizing. Mass spectra were acquired in the positive mode in the mass range from 100 to 1700 m/z. In the experiments described here, the mass spectrometer was operated in PASEF mode with exclusion of single charged peptides. A number of ten PASEF MS/MS scans were performed in 1.25 s from charge range 2–5.

Preview software (Byos TM, Protein Metrics, Cupertino, CA, USA) was employed to estimate the quality of the tryptic digestion in the samples and to predict the post-translational modifications present. The result was used for the “database search/identification” part. The sequence of the peptide was determined based on the fragmentation pattern. A search of an updated UniProt Homo sapiens database was performed in Peaks XPro software (Peaks XPro softwar Bioinformatic Solutions Inc., Waterloo, ON, Canada). The variable modifications allowed were as follows: K-acetylation, methionine oxidation, Deamidation (NQ), and Nerm-PyroGlu (Q). In addition, C-Propionoamide was set as fix modification. “Trypsin” was selected as Specific. Mass accuracy was set to 20 ppm and 0.05 Da for MS and MS/MS mode, respectively. Data were filtered according to an FDR of 5%. Protein redundancy was eliminated based on the presence of an identical set or subset of peptides.

To quantify the relative levels of protein abundance between different groups, samples were analyzed using the label-free quantification feature in the PEAKS XPro software. Feature detection was performed separately on each sample by the expectation-maximization-based algorithm. The features of the same peptide from all replicates of each sample were aligned through the retention time alignment algorithms. Mass error tolerance was set at 30 ppm, ion mobility tolerance (1/k0) at 0.07 and retention time tolerance at 7 min. Normalization factors of the samples were obtained by the total ion current (TIC) of each sample. The level of protein abundance was calculated as the summed area of the top three unique peptides. A significance $\ge$15 using ANOVA (p 0.05) as statistical method were used to determine those enriched proteins from the three compared conditions. (One or two accessions were significantly modulated from the three compared conditions). A heatmap was generated by the Spearman clustering method, using the ComplexHeatmap package in R. Enrichments in molecular process, cellular process and pathways (KEGG, Wikipathway and Reactome) were performed using ClueGo App from Cytoscape software (ver3.9.1, San Diego, CA, USA). ClueGO functional analysis was performed using GO BiologicalProcess EBI UniProt GOA-ACAP-ARAP (25.05.2022), GO MolecularFunction EBI UniProt GOA-ACAP-ARAP (25.05.2022) and KEGG (25.05.2022). Statistical Test Used = Enrichment/Depletion (Two-sided hypergeometric test), Correction Method Used = Bonferroni step down, Min GO Level = 3, Max GO Level = 8, Cluster #1, Sample File Name = File selection: ManuallyAddedOrModifiedIDs, Number of Genes = 3, Min Percentage = 4.0, GO Fusion = true, GO Group = true, Kappa Score Threshold = 0.4, Over View Term = SmallestPValue, Group By Kappa Statistics = true, Initial Group Size = 1, Sharing Group Percentage = 50.0.

Early-passage HUVECs from a single donor (#798) were a gift from Dr. N. Cherradi (Biology and Biotechnologies for Health Laboratory, CEA-Grenoble, France). For routine culture, HUVECs were passaged twice per week, seeded at 3500 cells/cm${}^2$ in M199 (Gibco® Medium 199 + GlutaMAX, Fischer Scientific, Illkirch, France) supplemented with 2% Supplements (Cascade Biologics LSGS 50X, Life Technologies, Fischer Scientific, Illkirch, France), 20% fetal bovine serum (FBS, Eurobio Scientific, Les Ullis, France) and 1% of penicillin-streptomycin antibiotics (Gibco®, Pen Strep, Fischer Scientific, Illkirch, France).

For bystander studies, conditioned media were obtained as described above, at 6 and 24 h after irradiation (or not) with X-ray or C-ions. To match HUVEC optimal culture conditions as closely as possible, the conditioned media were diluted by half using 2 X M199 medium. HUVEC cultures, after passages 5 to 8, were expanded for 3 days in 12-well plates and then incubated at 37 °C for 24 h in a humidified incubator with 5% CO2 without or with LPS (100 ng/mL, cat # L3024, Sigma-Aldrich, Saint-Quentin-Fallavier, France). Then supernatants were harvested to quantify cytokine secretion. Bystander HUVEC supernatants were centrifuged (10 min at 1400 rpm) and filtered to eliminate cells fragments, and aliquots were then frozen at –20 °C until use.

We simultaneously measured the secretion of IL-6, IL-8, IP-10, GM-CSF, MCP-1, VEGF and TNF$\alpha$ in supernatants of bystander HUVECs collected after 24 h of culture with conditioned media. We used a custom multiplex ELISA bead-based assay (LEGENDplex, Biolegend, OZYME, St Cyr L’Ecole, France), according to manufacturer’s instructions. Samples were run on a FACSCalibur flow cytometer (Becton-Dickinson Life Sciences-Biosciences, Le Pont-de-Claix, France), and results were analyzed with LEGENDplex v8.0 software (LEGENDplex, Biolegend, OZYME, St Cyr L’Ecole, France). Cytokine concentrations in supernatants were normalized on the number of live HUVECs in each condition. Results are presented as fold change compared to control conditions (HUVECs cultured in non-irradiated conditioned media).

The radiation-induced bystander effect was evaluated at the proteome level in bystander chondrocytes that had received the conditioned medium of chondrosarcoma cells irradiated with 0 Gy (control), 0.1 Gy or 2 Gy of X-ray or C-ions. The experiments were performed in biological triplicates. The medium-transfer protocol was selected for consistency with our previous characterization of bystander cells and their secretome and proteome [21, 29] (Supplementary Fig. 1). This protocol allowed us to compare the cell responses of non-irradiated cells (bystander response) across irradiation doses (0 vs. 0.1 vs. 2 Gy) and irradiation quality (X-ray vs. C-ions). Immediately after irradiation treatment, chondrosarcoma cells were cultured in fresh medium, which was itself never irradiated (Fig. 1). Chondrocytes were incubated for 24 h with the conditioned medium. Proteins from total cellular lysates of the chondrocytes were extracted and digested in solution, and the resulting peptides were analyzed using LC-MS/MS.

In total, 64,517 peptides, assembled into 6104 proteins, were identified from the 18 samples analyzed. Differential expression of proteins was considered significant when the p-value exceeded 0.05 (see Methods).

Control samples from X-ray and C-ion experiments were grouped together as biological replicates to reduce biological variability between experiments and to allow direct comparison between doses and irradiation qualities. These control samples corresponded to chondrocytes that had received conditioned medium from sham-irradiated (X-ray and C-ion) chondrosarcoma cells. Controls were compared with chondrocytes cells that had received conditioned medium from chondrosarcoma cells irradiated with X-rays (Comparison 1) or with C-ions (Comparison 2). Finally, a third comparison was performed between chondrocytes in medium conditioned by chondrosarcoma cells irradiated with X-rays (0.1 Gy) and chondrocytes in medium conditioned by chondrosarcoma cells irra diated with C-ions (0.1 Gy) (Table 1).

The first comparison identified proteome changes in bystander cells that had received medium from X-ray irradiated cells. A total of 179 accessions (protein groups) were found to be differentially expressed (p 0.05); 32 of these were highly significant (p 0.01) (Supplementary Fig. 2, Supplementary Table 1). Gene Ontology (GO) enrichment analysis of all protein groups identified impacted pathways (Fig. 2A). Globally, 19 groups of pathways were identified as significantly enriched (Supplementary Fig. 3, Supplementary Table 2). The first five GO enrichments corresponded to the formation of cytoplasmic translation initiation complex (27.42%), mitochondrial transport (11.29%), antioxidant activity (8.06%), glutamate and glutamine metabolism (6.45%) and gene and protein expression by JAK-STAT signalling after interleukin-12 stimulation (6.45%). Two additional GO enrichments were noteworthy: DNA duplex unwinding, including homologous recombination (3.23%) and positive regulation of double-strand break repair (1.61%).

Fig. 2.

ClueGO functional analysis (% terms per group) of the modulated accessions from. (A) The comparison 1 (0 Gy vs. X-ray 0.1 Gy vs. X-ray 2 Gy). (B) The comparison 2 (0 Gy vs. carbon ion 0.1 Gy vs. carbon ion 2 Gy). (C) The comparison 3 (0 Gy vs. X-ray 0.1 Gy vs. carbon ion 0.1 Gy). See the method section for the ClueGO settings.

The second comparison identified changes in the proteome of bystander samples that had received medium conditioned by C-ion-irradiated cells. A total of 126 accessions were found to be modulated (p 0.05); of these, 26 were highly significant (p 0.01) (Supplementary Fig. 4, Supplementary Table 1). GO enrichment analysis identified impacted pathways (Fig. 2B). Globally, 15 groups of pathways were identified as significantly enriched (Supplementary Fig. 5, Supplementary Table 3). The first five GO enrichments corresponded to L13a-mediated translational silencing of ceruloplasmin expression (30.3%), regulation of PTEN stability and activity (12.12%), proton-transporting ATPase activity, rotational mechanism (12.12%), mitochondrial transport (9.09%) and lamellipodium assembly (6.06%). Two additional GO enrichments were noteworthy: synaptic vesicle endocytosis (3.03%) and mitotic G2 DNA damage checkpoint signaling (3.03%).

The third comparison identified differences in the bystander effect after low-dose X-ray versus low-dose C-ions; the proteome of bystander samples in medium conditioned by cells irradiated by low-dose X-ray were compared to those in medium from cells irradiated by low-dose C-ions. A total of 182 accessions were found to be modulated (p 0.05); 41 of these were highly significant (p 0.01) (Supplementary Fig. 6, Supplementary Table 1). GO enrichment analysis identified impacted pathways (Fig. 2C). Globally, 12 groups of pathways were identified as significantly enriched (Supplementary Fig. 7, Supplementary Table 4). The first five GO enrichments corresponded to translation processes (Group 11, 43.08%), the separation of sister chromatids (Group 10, 16.92%), Huntington disease (Group 9, 9.23%), post-transcriptional regulation of gene expression (Group 8, 9.09%) and interleukin-12 signaling (Group 7, 4.62%). Two additional GO enrichments were noteworthy: positive regulation of the response to DNA damage stimulus (Group 5, 4.62%) and regulation of the EGF receptor signaling pathway (Group 1, 1.54%).

In parallel to the proteomic analysis, the same conditioned media were applied to HUVECs to look for an inflammatory response, potentially indicated by cytokine levels (Fig. 1). First, HUVECS were tested for their capacity to secrete several cytokines, with or without LPS activation (Supplementary Fig. 8). Treatment with LPS induced a significant increase in the concentration of all the cytokines tested (IL-8, IP-10, IL-6, MCP-1, GM-CSF, TNF$\alpha$ and VEGF). The quantification of these cytokines in the medium of HUVECs appeared reproducible and accurate enough to test the bystander effect.

Two independent experiments were performed for the two different radiation types, X-rays and C-ions. In each experiment, conditioned media from chondrosarcoma cells irradiated at different doses was applied to HUVECs, and the level of each cytokine was observed. The sample with sham-irradiated conditioned media was used to normalize measured cytokine levels.

Depending on the dose, the irradiation quality and the cytokine tested, a non-explained variability was observed between the two experiments (Supplementary Fig. 9). In the C-ion experiment, the levels of IL-8, IL-6, GM-CSF, MCP-1 and TNF$\alpha$ tended to decrease with increasing dose of radiation. This decrease was more pronounced in the absence of LPS activation (Supplementary Fig. 10). In the X-ray experiment, the opposite trend, with higher variability, was observed for IL-6, GM-CSF and TNF$\alpha$, again without LPS activation. This unexpected variability precluded further analysis of these results.

This study follows on a previous study of the bystander response in chondrocytes receiving conditioned medium from chondrosarcoma cells that had been irradiated with low-dose X-rays [29]. In the previous study, a gel-based analysis of the proteome of bystander cells indicated several potential effectors, pathways and mechanisms. These included oxidative stress response, cellular motility, exosome pathways and stress granules [29]. For the present study, we maintained the same protocol for cellular conditions [21, 29]. However, label-free mass spectrometry-based analysis allowed a deeper and broader investigation of the cellular response of bystander chondrocytes (Supplementary Fig. 10).

We observed that several GO pathways were enriched, including the cytoplasmic translation complex, mitochondrial transport, antioxidant activity and JAK-STAT signaling after interleukin-12 stimulation. Some of these pathways were also observed in our previous studies. For example, the interleukin-12 signaling pathway was proposed to be involved in the propagation of the bystander effect, through the modulation of cyclophylin A [29, 34]. Pathways connecting oxidative stress and translation complex stimulation proteins were impacted as well [29]. The modulated proteins were different, but the impacted pathways were similar.

Our study indicated that several pathways typically altered following direct irradiation of cells (i.e., homologous recombination and double-strand break repair) also experience bystander effects [10, 17, 20].

We constructed an in silico functional protein association network, using the program String, to reveal the connections between all modulated accessions (Fig. 3). Five processes with high probability of interaction emerged from this network, including the ribonucleoprotein granule (blue) and complex (green), cellular response to stress (yellow), the oxidation-reduction process (magenta) and the interleukin-12-mediated signaling pathway (red) (Table 2). We confirmed that stress granules were enriched [29] by a connection with several accessions that had previously been implicated in the cytoplasmic translation complex.

Fig. 3.

Full String network of the modulated accessions from the comparison 1 (0 Gy vs. X-ray 0.1 Gy vs. X-ray 2 Gy). The minimum interaction score was set to high confidence (0.700) score, and disconnected nodes were hidden in the network. The corresponding colour and GO were displayed in Table 2 (https://string-db.org/).

The radiation-induced bystander effect began rapidly, within several minutes after radiation [35]. The mechanism of the rapid cellular response likely involves stress granule formation and protein translation modulation.

We observed bystander effects on oxidative stress responses (or oxidation-reduction processes) with their 21 involved proteins (Table 2). Bystander activation of these responses has been observed in other model systems, recently in HepG2 and normal liver cells [36], human hematopoietic progenitor and stem cells [37] and mice exosomes [38].

The activation of JAK-STAT signaling after interleukin-12 stimulation was less expected. According to our proteomic study, the proteins MIF, AIP, BOLA2B, SOD2 and RPLP0 were modulated in bystander chondrocytes. These proteins are all involved in the interleukin-12-mediated signaling pathway. The interleukin-12 cytokine likely plays a central role in regulating immune responses, synergizing with several other cytokines for increased immune-regulatory activities in inflammation responses [39].

To further explore how signals from the bystander effect might influence the immune microenvironment, we also analyzed whether chondrosarcoma-conditioned culture media could modulate inflammatory signaling in HUVECs, as has been described for monocytes and macrophage cells. Our supernatant transfer experiments provide preliminary evidence that irradiated chondrosarcoma cells release soluble factors able to modulate the secretion of several cytokines from resting or LPS-stimulated HUVEC cells.

Some bystander responses of chondrocytes seem to be similarly stimulated across different types of irradiation. Initiation of cytoplasmic translation was the most pronounced GO enriched in the bystander response to C-ion irradiation and was likewise observed following X-ray irradiation. Change in mitochondrial transport was also observed after both kinds of irradiation.

In contrast, the regulation of intracellular pH and G1/S DNA damage checkpoints appeared to be bystander responses specific to C-ion irradiation. Modulation was observed in proteins PCBP4, PSMD12, PSME1 and XIAP, which are all involved in G1/S DNA damage response. This bystander response to C-ion irradiation has not been reported previously and could represent a specific signature of this biological effect. Some of these proteins were also associated with the extracellular exosome (Fig. 4, Table 2).

Fig. 4.

Full String network of the modulated accessions from the comparison 2 (0 Gy vs. carbon ion 0.1 Gy vs. carbon ion 2 Gy). The minimum interaction score was set to high confidence (0.700) score, and disconnected nodes were hidden in the network. The corresponding colour and GO were displayed in Table 2 (https://string-db.org/).

Other affected accessions were related to translation processes. Mitotic G2 DNA damage checkpoint signaling was also impacted, through proteins MRE11, TAOK1 and UIMC1. A modulation of cell cycle progression that would reduce protein translation could explain a decrease in cell division and motility that has been observed in other studies [21].

Finally, in our analysis of low-dose bystander response, we observed changes in a pathway that was related to the regulation of chromosome separation. This step in mitosis, between metaphase and anaphase, seemed to be impacted by low-dose irradiation with both X-rays and C-ions. Proteins such as BCL7C, BUB3, CENPF, DYNC1LI1, SMARCA4 or SMC4 were modulated. Some of these accessions are involved in separation and exchange of sister chromatids (Supplementary Table 4).

The induction of sister chromatid exchange has been observed in CHO cells irradiated in the G1 phase of the cell cycle with alpha-particles [40]. Although 30% of the cells showed an increased frequency of sister chromatid exchanges after exposure to 0.31 mGy, less than 1% of cell nuclei were actually traversed by an alpha-particle; this phenomenon was not observed in cells defective in homologous recombination [41]. In addition to such modification of chromosome separation, other of the pathways previously impacted by X-ray irradiation (translation, stress granules, extracellular vesicles, and interleukin-12) were still observed within the corresponding String network (Fig. 5 and Table 2).

Fig. 5.

Full String network of the modulated accessions from the comparison 3 (0 Gy vs. X-ray 0.1 Gy vs. carbon ion 0.1 Gy). The minimum interaction score was set to high confidence (0.700) score, and disconnected nodes were hidden in the network. The corresponding colour and GO were displayed in Table 2 (https://string-db.org/).

In interpreting the results of C-ion irradiation, one must consider the relative biological effects (RBEs), especially when comparing C-ions and X-rays [7, 42]. Our analyses included no correction factors, only physical doses of C-ions, as has been recently recommended [43]. Our biological samples were not directly irradiated with C-ions; bystander chondrocytes received only conditioned media from irradiated chondrosarcoma cells. In previous experiments of clonogenic survival, when a comparison of direct effect was performed with X-rays [32], a significant RBE was observed on chondrosaroma cells irradiated with C-ions (RBE ~3), depending on the cell lines and the LET of C-ions. When bystander cells were analyzed with the same approach, a reduction in clonogenic survival was observed at low doses of X-rays and C-ions. However, no RBE of C-ions was observed, as the effect was stronger with X-rays in comparison with C-ions [21]. For this reason, it seems unlikely that the proteomic differences observed in our study between X-rays and C-ions would be related to the RBE of C-ions observed when cells were directly irradiated. The observed differences were more likely related to a qualitative effect of C-ions, which induced different types of DNA damage, such as multiple localized breaks, that are more difficult repair for the cells [44]. Factors secreted by damaged cells could induce a differential response in bystander cells, when compared with X-ray irradiation, as observed in this study. If RBE-corrected doses were applied for C-ion irradiation, as is done during carbon therapy, this could modify the bystander response [45]. In our study, the bystander effect was observed at low dose (about 0.1 Gy). If an RBE-correction were applied, the dose would be divided by three, or about 30 mGy. Such an analysis would be interesting in the context of clinical C-ion therapy.

Irradiation stimulates a DNA damage response in the directly affected cells. It is now clear that cell-cell communication in the microenvironment of irradiated cells also plays a significant role in the cell- and tissue-level resроnse tо rаdiаtiоn exроsure. However, it is challenging to define the impact of this bystander effect in a clinical context because the extent of the effect depends on the tissue and the irradiation dose. Previous studies have reported possible impact of intercellular communication in the biological dose distribution of a treatment plan, with a signal range of 15 mm and an increased dose, up to 30% [46].

We observed bystander effects in non-cancerous cells at low doses of both X-ray and C-ions. This finding suggests important potential impact on nearby non-targeted regions. We report bystander effects on protein translation and oxidative stress homeostasis, which could alter normal cell functioning and potentially delay tissue reconstruction. Several factors involved in G1/S and G2 DNA damage checkpoints were affected, which could also reduce cells’ capacity to divide or to repair DNA damage.

C-ion irradiation is considered more accurate than X-ray irradiation, based on the physical properties of the ions. However, we observed bystander effects on DNA damage checkpoints following C-ion irradiation. Thus, the bystander effect could induced a kind of “biological inaccuracy” of C-ions, for which the magnitude is still to be defined. Our results indicate a necessity to consider the impact on normal tissue close to the tumor during C-ion therapy.

On the other hand, a modification of the microenvironment within the tumor bed could benefit to the treatment, by recruiting immune cells and/or promoting anti-tumor immunity [47]. C-ion irradiation has been associated with a possible increase in inflammation. In a mouse glioma model, C-ion irradiation induced an antiangiogenic and immune-permissive niche in association with a reduced viability of hypoxic and stem cell-like tumor cells [48]. Furthermore, in a human osteosarcoma cell model, an upregulation of PD-L1 expression after high-LET carbon-ion irradiation was greater than that induced by X-rays at the same biological dose [49]. However, such an inflammation response was not observed as the first mechanism involved in the C-ion-related bystander effect. Moreover, in our inflammation analysis on endothelial cells in medium from C-ion-irradiated chondrosarcoma cells, we observed a decline in the secretion of selected inflammatory cytokines.