Methods for studying the action of low-energy electron emissions on single cells

New approaches are needed for studying the radiobiological effects of low-energy electrons on living cells. This thesis describes the development, the validation, and the use of two new tools, called COOLER (COmputation Of Local Electron Release) and CoCoNut (Colony Counter developed by the Nutech department at the Technical University of Denmark).

COOLER is a general purpose absorbed dose calculation method that has been entrusted to a novel software program of the same name. It uses Monte Carlo simulations of monoenergetic point dose kernels as a base for finding analytical solutions to cellular dosimetry problems. The COOLER approach is applicable to a wide range of cellular geometries and the software handles electron energies up to 50 keV.

Electron ranges are calculated and compared to MIRD (Medical Internal Radiation Dose) predictions, whose results appear to be overestimated above 20 keV. Cellular S-values are obtained for dierent activity distribution scenarios and compared to MIRD predictions and Monte Carlo simulations. We prove that COOLER can successfully reproduce Monte Carlo results using an analytical approach.

By comparing COOLER to MIRD results, we show that the largest discrepancies between the two methods can be expected for electrons between 25 and 30 keV for a V79 cellular model. For those energies, S-values disagree from 50 to 100%, depending on the activity distribution. MIRD predictions fail the most when the activity is modeled on the cell surface.

Description of COOLER is contained in the publication: Siragusa et al. The COOLER code: a novel analytical approach to calculate subcellular energy deposition by internal electron emitters. Radiat Res. 2017;188(2):204-220. COOLER has been successfully employed to calculate absorbed dose values of free floating and attached V79 cells contaminated with tritiated water (HTO).

In order to use HTO as a model for investigating the biological effects of low-energy electrons, experiments have been carried out avoiding tritium incorporation into precursor biomolecules. In this thesis, we compare low-energy electron cell-killing efficacy to that of external photon irradiation using RBE (Relative Biological Effectiveness) values obtained from clonogenic cell survival experiments.

RBEs are calculated at the 10% survival fraction and are based on the ratio of COOLER-calculated absorbed doses. Irrespective of the cell geometry, RBE results for floating and adherent cells are always 2. Results confirm that low-energy electrons contribute significantly more to the radiation damage than could be expected for such electrons.

Moreover, COOLER shows that the change in the cell culture growth condition is relevant as suspended V79 cells tend to quickly form small cell clusters, which are responsible for an increased radiation resistance of the cells. The role of the dose rate of the reference radiation for RBE-calculations is also investigated.

In our experiments, RBEs range from 1.6 to 2.0 for adherent V79 cells, when compared respectively to acute exposures or to similar dose rates of external g-rays. Results on HTO experiments have been published: Siragusa et al. Radiobiological effects of tritiated water short-term exposure on V79 clonogenic cell survival.

Int J Radiat Biol. 2018;94(2):157-165. All radiobiological results contained in the previously mentioned article have been obtained counting many cell clones in a number of clonogenic cell survival experiments. In this thesis, I describe the development of a combined software/hardware tool, called CoCoNut, that automates the otherwise slow counting process.

CoCoNut consists of an open source ImageJ macro and a 3D-printable photographic light-box, engineered to work together. The full method is tested against V79 cell survival in cell culture flasks and Petri dishes. It proves able to identify cell clones with unconventional morphology, to successfully distinguish between single and merged colonies, and to identify colonies bordering on flask edges.

Description of CoCoNut is given in the study: Siragusa et al. Cell colony counter called CoCoNut. PLoS One. Manuscript number: PONE-D-18-03194. Under preparation for resubmission after initial review. Finally, COOLER is used to compute accurate absorbed dose calculations for the novel Auger electron emitter 135La, as reported in the publication: Fonslet et al. 135La as an Auger-electron emitter for targeted internal radiotherapy". Phys Med Biol. 2017;63(1):015026.

In that article, COOLER-derived cellular S-values are compared to MIRD results for a spherical V79 cellular model. S-value contributions to the cell nucleus from different source regions are determined. When compared to MIRD, COOLER S-value results show an increased dose for the combinations N Cy (38%) and N CS (89%), while the N N case shows a 5% decrease.

Being N the cell Nucleus, Cy the Cytoplasm, and CS the Cell Surface. Our results make clear that MIRD calculations may underestimate absorbed dose values for tumor treatment plannings based on 135La.  COOLER and CoCoNut have succeeded in giving meticulous and prompt radiobiological results for our experiments.

Therefore, their combination is suitable for future research projects aimed at assessing the role of low-energy electrons in, for example, therapeutic applications and radiation protection scenarios.