Evaluation of in vitro irradiation setup: Designed for the horizontal beamline at the Danish Centre for Particle Therapy

Background Radiobiological experimental setups are challenged by precise sample positioning along depth dose profile, scattering conditions, and practical difficulties that must be addressed in individual designs. The aim of this study was to produce cell survival curves with several irradiation modalities, by using a setup designed at the Danish Centre for Particle Therapy (DCPT) for in vitro proton irradiations using a horizontal beam line and thereby evaluating the setups use for in vitro irradiations experiments. Materials and methods The setup is a water phantom suitable for in vitro research with multiple irradiation modalities, in particular the pencil scanning proton beam available from a horizontal experimental beamline. The phantom included a water tank of 39.0 × 17.0 × 20.5 cm. Cell survival-curves were produced using the cell line V79 Chinese hamster lung fibroblast cells (V79s) in biological triplicates of clonogenic assays. Cell survival curves were produced with both a 18 MeV electron beam, 6 MV photon beam, and a Spread-Out Bragg Peak (SOBP) proton beam formed by pristine energies of 85–111 MeV where three positions were examined. Results Survival curves with uncertainty areas were made for all modalities. Dosimetric uncertainty amounted to, respectively, 4%, 3% and 3% for proton, electron, and high energy photon irradiations. Cell survival fraction uncertainty was depicted as the standard deviation between replications of the experiment. Conclusion Cell survival curves could be produced with acceptable uncertainties using this novel water phantom and cellular laboratory workflow. The setup is useful for future in vitro irradiation experiments.


Appendix 3 -Details on cell conditions
For all experiments V79s were cultured in Thermo Scientific TM Nunc TM T75 culture flasks and transferred to Thermo Scientific TM Nunc TM T25 flasks for irradiation aiming for 120 colonies after the final incubation period in the experimental samples.These flasks were equipped with filter-caps that ensured 5% CO2 within the flasks while preventing contamination when removed from the incubator.
Samples were then incubated for 6 to 8 hours in horizontal position prior to irradiation.The same culture medium (MEM, 10% FBS, 1% p/s, 1% NEAA, 1% SP) was used throughout the experiments.Colonies were counted with the standard threshold of 50 cells per colony.After counting all irradiated flasks, survival fractions (SF) were calculated on each individual flask using Equation 1: Equation 1: Survival fractions. was obtained colonies in irradiated flasks normalized to seed out volume. was average count of cells in the 6 controls (reference flask on each biological replicate) normalized to seed out volume in controls.

Appendix 4-Irradiation techniques
LINAC 6 MV photons and 18 MeV electrons LINAC 6 MV photons and 18 MeV electrons irradiations were caried out using a 100 cm source-to-axis distance TrueBeam linear accelerator (Varian Medial Systems, Palo Alto, USA).The setup was CT scanned (Brilliance CT BigBore, Philips Medical Systems, Cleveland, USA) with a 0.5 mm slice thickness and imported for treatment planning.A reference point was chosen to coincide with the centre of mass of the cell-layer, and the number of monitor units (MU) needed to deliver a certain dose was calculated by normalizing the treatment plan to deliver 100% prescribed dose in the reference point.For 6 MV photon irradiations, the cell layer was positioned 2.0 cm from the outer wall as measured along the beam direction, and the treatment was planned with a gantry rotation of 90 degrees, a 10 cm x 10 cm jaw-defined field, 100 cm source-surface-distance (SSD), a flattened 6 MV beam quality and a machineset dose rate of 600 MU/min corresponding to a dose rate at the cells of approximately 6.4 Gy/min.For electron irradiations, the cell layer was positioned 2.3 cm from the outer wall as measured along the beam direction and the treatment was planned with a gantry rotation of 90 degrees, a 10 cm x 10 cm aperture-defined field, an SSD of 97.7 cm, an 18 MeV beam quality and a machine-set dose rate of 600 MU/min corresponding to a dose rate at the cells of approximately 6.3 Gy/min.Beam quality and distance from outer wall were chosen to optimize robustness of the setup and dose uniformity.Prior to conducting the experiments, the machine output factor was determined following institutional guidelines using a farmer chamber (FC65-G, IBA Dosimetry GmbH, Schwarzenbruck, Germany) and the number of MU needed to deliver a certain dose was corrected accordingly.The setup was aligned with the cell layer in sagittal orientation and centered in the field using the in-room treatment lasers by measuring the distances, as given from the TPS, from the sagittal, transversal, and frontal lasers (supine, head-first coordinate system) to the outer walls of the phantom.Samples received the prescribed dose (as seen in appendix 6) with a total dose uncertainty of +/-3% including calibration uncertainties of measurement chambers.Pictures of the setup, and the simulated plan can be found in Appendix 7.

111-85 MeV Protons.
Proton irradiations were performed at the experimental fixed horizontal proton beamline with pencil beam scanning at The Danish Centre for Particle Therapy (DCPT) (ProBeam, Varian, a Siemens Healthineers company, Palo Alto, CA, USA).The beam energy range was 111-85 MeV.Flasks were placed in the water, with the cell layer at the water-equivalent depth of 3.2 cm (proximal), 7.7 cm (middle SOBP), or 8.2 cm (semi-distal SOBP).Flasks were irradiated one at a time, with the flask wall positioned in the middle of 9x9 cm 2 uniform field (+-1%).The alignment of the flask relative to the isocenter was verified by visual inspection of irradiated EBT-3 gafchromic film taped to the flask wall.
Prescribed doses were verified with the use of Roos-and Advanced Markus-ionization chambers, taped inside an empty cell flask (rail with flask containing Advanced Markus ionization chamber was aligned without fixating the slider with screws to match the reference point of Roos).The overall uncertainty of prescribed doses was found to be below 4% (not shown on the plot).Flasks were placed so that the beam entered the flask bottom, hit the cells, and continued through the cell medium.This was done under the assumption that the flask plastic varied less between flasks and between biological replicates, than the density of cell medium.Therefore, this methodology was chosen to ensure that cells received the same dose despite daily variations in the cell medium.The setup is seen in Appendix 7.

Appendix 5 -Cellular cooperation results
The curve is an exponential fit based on equation 1. Error bars are displaying SD of three technical replicates.