The response of normal tissues to synchrotron microbeam radiotherapy

© 2019 Jessica Audrey VenturaThis thesis presents multidisciplinary and collaborative research aimed at translating microbeam radiation therapy (MRT) to clinical trials. MRT is a novel pre-clinical synchrotron-based radiation therapy modality that is a potential substitute for conventional radiotherapy (CRT) modalities due to its improved therapeutic index facilitated by remarkable tolerance of normal tissues with successful tumour ablation. One of the main limitations of CRT is that while tumour ablation is dependent on radiation dose, the maximum tumour dose is limited by the radiation tolerance of the surrounding healthy normal tissues. A key attribute of MRT is the normal tissue sparing effect that is thought to occur due to the physical geometry of the MRT beam. MRT involves synchrotron-generated X-rays that are spatially fractionated to generate an array of wafer-thin parallel microbeams, known as peaks, separated by hundreds of microns, known as valleys. The valleys typically receive 2-4% of the peak dose. Presently there is no conclusive evidence that can fully explain the efficacy of MRT compared to synchrotron BB radiotherapy (a radiation modality that resembles CRT). Various hypotheses have been proposed to explain MRT effects including: radiation- induced bystander/abscopal effects (RIBE/RIAE), microvascular effects, increased tumour cell migration, increased cell proliferation and stem cell survival in normal tissues, and changes in gene regulation and immune response in tumours and normal tissues. My contribution in our multidisciplinary group is to investigate the biological mechanisms associated with synchrotron MRT. To establish if radiation parameters influence the RIAE, a range of variables including beam modality, dose and size of irradiation field were tested in vivo at 1 and 4 days post irradiation following local synchrotron X-ray BB and MRT. DNA damage, apoptosis and local and systemic immune responses were monitored in directly irradiated tissue and out-of-field tissue. We demonstrated that irradiation dose and size of irradiation field do not substantially influence RIAE in out-of-field tissues and a short pulse of MRT and BB irradiation was sufficient to induce persistent systemic and genotoxic effects in mice. While immune responses have been proposed as a possible mechanism that facilitates RIAE, it has never been demonstrated before. To identify which immune response components are involved in RIAE propagation, various RIAE endpoints (including DNA damage and apoptosis) were tested in a range of out-of-field tissues of immune-deficient mice treated with MRT. We demonstrated that the abscopal effect relies on a functional immune response, particularly the involvement of macrophages, monocyte chemotactic protein 2 (CCL2) and TGFβ in order for RIAE to be transmitted to out-of-field tissues. Due to the complex dose profile of MRT, MRT dosimetry is problematic using current physical dosimeters. An alternative is to use γH2AX (a biomarker of DNA damage) as a biodosimeter to measure the biologically equivalent valley dose following MRT. A range of BB doses were used to generate a BB standard curve, which was used to extrapolate the biologically equivalent valley dose of a range of MRT peak doses. The results revealed that γH2AX may not be an appropriate biodosimeter to predict the biological valley dose of MRT due to the non-linear dose response observed in MRT- irradiated mouse skin and human fibroblast cells. The response of gastrointestinal tract (GIT) to MRT was also investigated since no normal toxicity data for GIT response to MRT exists. We investigated alterations in gene expression of 6 radiation responsive genes at 4 and 48 hrs post-irradiation in male and female mice using qRT-PCR following partial body irradiation of the abdomen using MRT and BB. The results showed that MRT and BB are able to induce significant modulations in gene expression in directly irradiated GIT tissues. MRT may induce more oxidative stress and reduce macrophage infiltration in GIT compared to BB, due to the significant downregulation of inflammation and DNA repair genes. Both modalities induced a protective response in the GIT by upregulating MDM2 and IL-10 at 4 hrs post-irradiation. MDM2 was most abundant in the colon compared to the upper GIT tissues. Unlike MRT, BB induced differential inflammation response via NFκβ2 expression in female mice compared to male mice, indicating that gender-specific mechanisms may play a role in response to BB irradiation. These studies provide further information on the biological mechanisms that contribute to the normal tissue sparing effects of MRT and could assist in optimisation of MRT treatment protocols. Further knowledge in mitigating or reducing normal tissue toxicities and increasing tumour control would greatly benefit cancer patients that require radiation therapy