An in vitro investigation of the impact of radiation induced bystander effect on the therapeutic irradiation of a prostate cancer cell line.

Abstract

Introduction. The aim of radiotherapy, in general, is to deliver a high enough radiation dose to tumour cells to control (and stop) their growth without causing severe complications to surrounding healthy tissues. As a result, it is very important to define a precise irradiation target for radiotherapy treatment. For many years only DNA has been seen as the main target for radiation to cause cellular death in living tissues. In the last decade the fundamental dogma of radiobiology, known as the ‘target theory’, has been reviewed. The extensive experimental evidence demonstrates that not only cell nucleus but also cellular cytoplasm, membrane, and even neighbouring cells, located outside the radiation field, should be viewed as possible targets for therapeutic ionising radiation. Methodology. The research described in this thesis aims to investigate the impact of the non-targeted effects of 6MV x-rays during the radiotherapy. This thesis intends to analyse the published mathematical models which predict occurrence and magnitude of radiation induced bystander effects (RIBEs), with experimental validation of one of these models. The methodology undertaken involved: • Literature review and development of comprehensive understanding of general concepts of radiation induced bystander effects; • Establishment of a suitable experimental methodology to investigate these phenomena, in particular radiation induced additional killing, in the application to radiotherapy to PC3 human prostate epithelial adenocarcinoma cell line, including: • evaluation of biological characteristics such as population doubling time and plating efficiency; • evaluation of radiobiological characteristics such as the dose which kills half of clonogenes (D₅₀), which will be used subsequently as the prescribed dose in the dose cold spot experiment; (in the experiment investigating cell survival in the under-dosed region) • determination of suitable biological end-points (such as apoptotic cell death, reduced proliferation rate, clonogenic cell death) following radiation treatment; • design of a dose-cold spot experiment to investigate RIBE in a reduced dose region (ie receiving ~80% of the prescribed dose) in freely communicating cells and non-communicating cells; • Investigation of the extent of non-targeted effects on cell killing in a dose cold spot in human prostate PC3 cancer cell line; • Analysis of RIBE related models; • Validation of the published stochastic model that relates absorbed dose to the emission and processing of cell death signals by non-irradiated cells which included: • determination of magnitude of medium-borne signals (affecting non-targeted cells) dependence on the radiation doses received by donor cells • investigation of donor cell concentration impact on the emission of death signals predicted by the model. All cell irradiations were performed at the Royal Adelaide Hospital, Radiation Oncology Department using a 6 MV x-ray beam produced by a Varian linear accelerator (Varian, Palo Alto, CA,USA). A clinically applied nominal dose rate of 3 Gy/min was used. Each radiation treatment was performed at 100 cm from the beam focal spot with 20 x 20 cm² radiation field size. The culture dishes were placed on the top of 1.5 cm thick solid water build up sheets. To avoid irradiation through air gaps cells were treated posteriorly with gantry positioned at 180°. Custom made wax phantoms (for different flask sizes) were used in conjunction with 5 cm thick solid water slab to cover the flask to ensure full scatter conditions. Machine radiation output was routinely checked with Daily QA 3™ device (Sun Nuclear, USA) before each radiation treatment. The primary research objectives were investigated through a series of research papers. Results. The findings and results of the experiments designed and performed in the current work include: I. Biological characteristics of PC3 cell line such as plating efficiency and population doubling time were found to be 0.60, 48 hours respectively. II. The fraction of cells surviving the standard clinical daily dose of 2 Gy (SF2) typical of curative radiation protocols was found to be 0.586 (± 0.0279), while the dose that killed half of the clonogen population (D₅₀) was found to be 2.037Gy. III. Radiosensitivity of PC3 cells differs widely among laboratories - the maximum difference found was 131.58%. This cell line appeared to be very sensitive to the methods used therefore it was important to evaluate D₅₀ independently rather than relying on published data. IV. Apoptotic assay revealed no significant dose dependant early cell deaths until 96 hours after radiation exposure. Following this time the first sizable colonies can be detected by the clonogenic survival assessment. Hence cellular damage in a dose cold spot was assessed by long term survival data which includes all types of radiation induced damages. V. Cells exposed to a dose cold spot that are freely communicating versus non-communicating cells revealed significant decrease (16.2%) in cells survival presumably due to intercellular communication. Validation of the stochastic model predicting emission and processing of cell death signals in non-irradiated cells revealed significant decreases in cell survival (P<0.001) exposed to irradiated cell condition media (ICCM) derived from donor cells of various concentrations and irradiated with different doses. Dependency of the toxicity of ICCM on the cellular concentration of donor cells was fond to be significant (p<0.5) as well. Conclusion. For the given cell line under existing growing and treatment conditions the cell survival in the dose cold spot region was significantly lower when under-irradiated cells were in contact with the cells receiving 100% of the prescribed dose compared to the cellular survival obtained from the under-dosed cells, by the same amount of radiation, which were treated separately. Presumably these variations were mainly due to intercellular communication. Significant reduction in PC3 cell survival after receiving ICCM was observed. Data fitting revealed an exponential decrease in recipient cell survival with the dose received by the ICCM. However the current experiment was not able to identify the associated dose threshold for the reduction in survival from ICCM due to the saturation of the effect at the doses investigated. This can be attributed to either saturation in signal generation due to limited signal potency or saturation in recipient cell responses. It appeared that death signal emission may increase with increasing numbers of radiation hits to a certain target and with increasing number of targets able to emit death signals. However, the effect saturates when it reaches a specific value in a number of hits or in an amount of critical targets. The mechanisms behind radiation induced additional killing are not clear yet. Little is known about the types of DNA damage affecting bystander cells. The impact of RIBEs in application to novel radiotherapy treatment techniques, such as intensity modulated radiation therapy and tomotherapy, needs further investigation as they deliver highly conformal doses to tumours, but cover bigger volumes with the low doses where bystander responses are more pronounced. Incorporation of RIBEs into the research that underpins clinical radiotherapy will result in a shift beyond simple mechanistic models currently used towards a more systems-based approach. It is a difficult task to design a coherent research strategy to investigate the clinical impact of bystander phenomena, given the complex protean nature of it. Any consideration of bystander effects will challenge clinicians' preconceptions concerning the effects of radiation on tumours and normal tissues and therefore disease management.