Artiste

Cells were exposed to IR using the linear accelerator Artiste™ (Siemens, Munich, Germany) (6-MV photons; dose-rate 2 Gy/min). Cells were analyzed at different time-points (5 h, 24 h, 1 w and 2 w post-IR) following IR (20 Gy) and compared to non-irradiated controls (non-IR).

Foreskin explants were rinsed with PBS and divided into small pieces (0.5 cm²). Foreskin samples were incubated dermal side down on polyethylene membranes and epidermal side exposed to air in 6-well plates (each well filled with 2 mL medium) at 37 °C under 5% CO₂. The culture medium consisted of DMEM, 10,000 units of penicillin, 10 mg/mL streptomycin, and 200 mM/L glutamine. After air-medium interface cultivation for 24 h, foreskin explants were exposed to IR with 10 Gy (6-MV photons, 2 Gy/min) using the linear accelerator Artiste™ (Siemens, Munich, Germany). Twenty-four hours after IR exposure, foreskin tissue samples were embedded for microscopic analysis, and the supernatant was used for cytokine measurement (Supplementary Figure S1A). Histopathological studies confirmed the viability and functionality of our foreskin culture method (Supplementary Figure S1B). Moreover, for different culture periods, we observed no variations in H2A.J+ cell numbers, suggesting that foreskin keratinocytes maintain their epigenetic status (Supplementary Figure S2).

Skin explants were divided into sections and rinsed with PBS. Skin sections were incubated dermal side down on polyethylene membranes and epidermal side exposed to air in six-well plates (each well filled with 2 ml medium) at 37°C, under 5% CO₂. The culture medium consisted of Dulbecco’s Modified Eagle Medium supplemented with 10% fetal calf serum, 10,000 units penicillin, 10 mg/ml streptomycin, and 200 mM/l glutamine. After air-medium interface cultivation for 12 hours, skin sections were exposed to IR with 10 Gy (6-MV photons, 2 Gy/min) using the linear accelerator Artiste™ (Siemens, Munich, Germany). 48 h after IR exposure, cultured epidermis sections were harvested for paraffin embedding and subsequent IFM analysis. Histopathological studies confirmed the viability and functionality of our skin organ culture method.

Eight-week-old male C57BL/6 mice (Charles River Laboratories, Sulzfeld, Germany) were housed in groups in IVC cages under standard laboratory conditions. Whole-body irradiation with 5 fractions of 2 Gy (daily IR exposure from Monday to Friday) was performed at the linear accelerator (Artiste™, Siemens), as described previously [24 (link)]. Then, 72 h, 1, and 2 weeks after the last IR exposure (sham-)irradiated animals (n = 3 per time-point and treatment-group, 18 animals in total) were anesthetized intraperitoneally using Ketamine and Rompun prior to tissue collection. Experimental studies were approved by the Medical Sciences Animal Care and Use Committee of Saarland.

Two computed tomography (CT) simulators (GE lightspeed 16, GE Healthcare, Chicago, IL, USA and Somatom Definition, Siemens Healthcare, Erlangen, Germany) equipped with moveable lasers (LAP GmbH, Germany) are located within our department. Both scanners are used for simulation and with no specific patients directed to either scanners. Eclipse (version 15.5, Varian Medical Systems, Palo Alto, CA, USA) is used as both a virtual simulator and TPS. The algorithms used for dose calculation are Acuros version 15.5 for photon and electron Monte Carlo (EMC) version 15.5 for electrons. The TPS is complemented by a software package (MIM Vista, version 2.6, MIM, OH, USA), which is capable of performing functions such as image fusion, contouring, and dose summation. An Oncology Information System (OIS), (Mosaiq, version 2.6, Elekta, CA, USA) is used to transfer plan parameters required for treatment delivery to six linear accelerators: four Truebeams, one clinac iX (Varian Medical Systems, Palo Alto, CA, USA), and one Artiste (Siemens Healthcare, Erlangen, Germany). All linear accelerators are equipped with conebeam CT and the Artiste is equipped with CT on rails.

MV CBCT images were acquired on a Siemens Artiste that was equipped with the In‐Line kView system. Instead of using the treatment beam, it used a low MV energy and a carbon target during imaging to improve image contrast.⁽¹⁴⁾ Images were acquired using between 5 and 15 monitor units (MUs) (about 5‐15 cGy per acquisition). Imaging protocols were chosen based on the phantom size. For small objects (size of a head), the regular field of view (rFOV) was sufficient to capture the entire anatomy and the system used a half‐arc of 200°. For larger objects (the size of a thorax or pelvis), the extended field of view (eFOV) was used. This mode used a 5.5 cm lateral flat‐panel offset during the acquisition and a 360° gantry rotation. Similar to the other machines, one of three different imaging protocols was chosen based on the treatment site.