Optimizing synchrotron microbeam radiotherapy for cancer treatment

© 2015 Dr. Mohammad Johari IbahimSynchrotron microbeam radiation therapy (MRT) is a novel, preclinical form of treatment that has proven effective at ablating tumours in a range of animal models, with significantly reduced damage to adjacent normal tissues. The cellular mechanisms by which MRT achieves this superior outcome are currently unknown. Several hypotheses have been proposed including radiation-induced bystander effects, altered cellular communication, micro-vascular effects and differential gene regulation related to the local tumour immune response. Synchrotron MRT is characterized by a very high in beam entrance dose (300 – 1000 Gy) and a valley dose of approximately 2% of the peak dose. The physical dosimetry associated with MRT is more complex than that of conventional broadbeam radiotherapy (BB) due to the different components of the MRT dose profile. To implement synchrotron MRT clinically for cancer treatment, it was first necessary to undertake dose equivalence studies to identify MRT doses that give similar outcomes to BB treatments. Current clinical radiotherapy protocols to determine radiation dosimetry that have been used for conventional radiotherapy are not applicable for synchrotron MRT. Clonogenic assay remain the gold standard cell-based technique for detecting radiobiological and radiation-induced bystander effects (RIBE) although these have several limitations. The xCELLigence Real Time-Cell Impedance System (RT-CIS) uses a non-invasive and label free method to create a cell index that is a composite measure of cell proliferation, metabolism and migration. Several studies have shown the xCELLigence RT-CIS can be used to measure radiobiological effects and in our hands the results of the impedance assay agreed with the clonogenic data. The aims of this study were to (i) evaluate novel and established assays to study radiobiological and radiation-induced bystander effects in an in vitro model, (ii) to undertake in vitro and in vivo studies to determine dose equivalence between BB and MRT by using novel and established assays, and (iii) to compare gene expression regulation in an in vivo tumour model irradiated with MRT or BB. Results obtained showed that xCELLigence RT-CIS can be used to measure radiation dose effects and these correlated with colonogenic assays. The xCELLigence RT-CIS and clonogenic assays can be used to estimate biological dose equivalence between MRT and BB in vitro using a sigmoidal dose response curve or linear quadratic model. The valley dose for MRT calculated using Monte Carlo simulation is not biologically equivalent to the BB biological dose, because the MRT valley dose alone does not determine the acute cellular response to MRT, which also varies between cell lines. High dose MRT (560 Gy) significantly up-regulated several genes (Ear11, Ccl24, Ccl6 and Ccl9) related to eosinophil function and recruitment in BALB/C mice inoculated with EMT6.5 tumour cells. However no difference in the accumulation of eosinophils in the tumour micro-environment between MRT and BB irradiated tumours was found by 48 hours post-irradiation. This thesis provides the first determination of biological dose equivalence between BB and MRT modalities for different cell lines and identifies xCELLigence RT-CIS assay as a suitable substitute for clonogenic assay for measuring radiation-induced biological effects. The results on biological dose equivalence will be useful for the safe selection of MRT doses for future veterinary and clinical trials. Furthermore, MRT differentially up-regulated eosinophil-related gene pathways compare to BB at doses previously demonstrated to be biologically equivalent, however no differences in eosinophil populations were detected in the tumour microenvironment by 48 hours post irradiation