Academic literature on the topic 'Radiation induced bystander effect; non-targeted effect in radiotherapy'

Radiation therapy is widely used as the primary treatment option for several cancer types. However, radiation therapy is a nonspecific method and associated with significant challenges such as radioresistance and non-targeted effects. The radiation-induced non-targeted effects on nonirradiated cells nearby are known as bystander effects, while effects far from the ionising radiation-exposed cells are known as abscopal effects. These effects are presented as a consequence of intercellular communications. Therefore, a better understanding of the involved intercellular signals may bring promising new strategies for radiation risk assessment and potential targets for developing novel radiotherapy strategies. Recent studies indicate that radiation-derived extracellular vesicles, particularly exosomes, play a vital role in intercellular communications and may result in radioresistance and non-targeted effects. This review describes exosome biology, intercellular interactions, and response to different environmental stressors and diseases, and focuses on their role as functional mediators in inducing radiation-induced bystander effect (RIBE).

Indirect effects may contribute to the efficacy of radiotherapy by sterilizing malignant cells that are not directly irradiated. However, little is known of the influence of indirect effects in targeted radionuclide treatment. We compared γ-radiation-induced bystander effects with those resulting from exposure to three radiohaloanalogues of meta-iodobenzylguanidine (MIBG): [131I]MIBG (low linear energy transfer (LET) β-emitter), [123I]MIBG (high LET Auger electron emitter), and meta-[211At]astatobenzylguanidine ([211At]MABG) (high LET α-emitter). Cells exposed to media from γ-irradiated cells exhibited a dose-dependent reduction in survival fraction at low dosage and a plateau in cell kill at > 2 Gy. Cells treated with media from [131I]MIBG demonstrated a dose-response relationship with respect to clonogenic cell death and no annihilation of this effect at high radiopharmaceutical dosage. In contrast, cells receiving media from cultures treated with [211At]MABG or [123I]MIBG exhibited dose-dependent toxicity at low dose but elimination of cytotoxicity with increasing radiation dose (i.e. U-shaped survival curves). Therefore radionuclides emitting high LET radiation may elicit toxic or protective effects on neighboring untargeted cells at low and high dose respectively. We conclude that radiopharmaceutical-induced bystander effects may depend on LET and be distinct from those elicited by conventional radiotherapy.

Studies have been conducted at synchrotron facilities in Europe and Australia to explore a variety of applications of synchrotron X-rays in medicine and biology. We discuss the major technical aspects of the synchrotron irradiation setups, paying specific attention to the Australian Synchrotron (AS) and the European Synchrotron Radiation Facility (ESRF) as those best configured for a wide range of biomedical research involving animals and future cancer patients. Due to ultra-high dose rates, treatment doses can be delivered within milliseconds, abiding by FLASH radiotherapy principles. In addition, a homogeneous radiation field can be spatially fractionated into a geometric pattern called microbeam radiotherapy (MRT); a coplanar array of thin beams of microscopic dimensions. Both are clinically promising radiotherapy modalities because they trigger a cascade of biological effects that improve tumor control, while increasing normal tissue tolerance compared to conventional radiation. Synchrotrons can deliver high doses to a very small volume with low beam divergence, thus facilitating the study of non-targeted effects of these novel radiation modalities in both in-vitro and in-vivo models. Non-targeted radiation effects studied at the AS and ESRF include monitoring cell–cell communication after partial irradiation of a cell population (radiation-induced bystander effect, RIBE), the response of tissues outside the irradiated field (radiation-induced abscopal effect, RIAE), and the influence of irradiated animals on non-irradiated ones in close proximity (inter-animal RIBE). Here we provide a summary of these experiments and perspectives on their implications for non-targeted effects in biomedical fields.

Metal nanoparticles are effective radiosensitizers that locally enhance radiation doses in targeted cancer cells. Compared with other metal nanoparticles, gold nanoparticles (GNPs) exhibit high biocompatibility, low toxicity, and they increase secondary electron scatter. Herein, we investigated the effects of active-targeting GNPs on the radiation-induced bystander effect (RIBE) in prostate cancer cells. The impact of GNPs on the RIBE presents implications for secondary cancers or spatially fractionated radiotherapy treatments. Anti-prostate-specific membrane antigen (PSMA) antibodies were conjugated with PEGylated GNPs through EDC–NHS chemistry. The media transfer technique was performed to induce the RIBE on the non-irradiated bystander cells. This study focused on the LNCaP cell line, because it can model a wide range of stages relating to prostate cancer progression, including the transition from androgen dependence to castration resistance and bone metastasis. First, LNCaP cells were pretreated with phosphate buffered saline (PBS) or PSMA-targeted GNPs (PGNPs) for 24 h and irradiated with 160 kVp X-rays (0–8 Gy). Following that, the collected culture media were filtered (sterile 0.45 µm polyethersulfone) in order to acquire PBS- and PGNP- conditioned media (CM). Then, PBS- and PGNP-CM were transferred to the bystander cells that were loaded with/without PGNPs. MTT, γ-H2AX, clonogenic assays and reactive oxygen species assessments were performed to compare RIBE responses under different treatments. Compared with 2 Gy-PBS-CM, 8 Gy-PBS-CM demonstrated a much higher RIBE response, thus validating the dose dependence of RIBE in LNCaP cells. Compared with PBS-CM, PGNP-CM exhibited lower cell viability, higher DNA damage, and a smaller survival fraction. In the presence of PBS-CM, bystander cells loaded with PGNPs showed increased cell death compared with cells that did not have PGNPs. These results demonstrate the PGNP-boosted expression and sensitivity of RIBE in prostate cancer cells.

The radiation-induced bystander effect (RIBE), an important non-targeted effect of radiation, has been proposed to be associated with irradiation-caused secondary cancers and reproductive damage beyond the irradiation-treated area after radiotherapy. However, the mechanisms for RIBE signal(s) regulation and transduction are not well understood. In the present work, we found that a Golgi protein, GOLPH3, was involved in RIBE transduction. Knocking down GOLPH3 in irradiated cells blocked the generation of the RIBE, whereas re-expression of GOLPH3 in knockdown cells rescued the RIBE. Furthermore, TNF-α was identified as an important intercellular signal molecule in the GOLPH3-mediated RIBE. A novel signal axis, GOLPH3/ERK/EGR1, was discovered to modulate the transcription of TNF-α and determine the level of released TNF-α. Our findings provide new insights into the molecular mechanism of the RIBE and a potential target for RIBE modulation.

Salivary glands sustain collateral damage following radiotherapy (RT) to treat cancers of the head and neck, leading to complications, including mucositis, xerostomia and hyposalivation. Despite salivary gland-sparing techniques and modified dosing strategies, long-term hypofunction remains a significant problem. Current therapeutic interventions provide temporary symptom relief, but do not address irreversible glandular damage. In this review, we summarize the current understanding of mechanisms involved in RT-induced hyposalivation and provide a framework for future mechanistic studies. One glaring gap in published studies investigating RT-induced mechanisms of salivary gland dysfunction concerns the effect of irradiation on adjacent non-irradiated tissue via paracrine, autocrine and direct cell–cell interactions, coined the bystander effect in other models of RT-induced damage. We hypothesize that purinergic receptor signaling involving P2 nucleotide receptors may play a key role in mediating the bystander effect. We also discuss promising new therapeutic approaches to prevent salivary gland damage due to RT.

Abstract Radiation-induced neurocognitive dysfunction (RIND) has attracted a lot of attention lately due to the significant improvement of the survival of cancer patients after receiving cranial radiotherapy. The detailed mechanisms are not completely understood, but extensive evidence supports an involvement of the inhibition of hippocampal neurogenesis, which may result from radiation-induced depletion of neural stem cells (NSCs) as well as the damage to neurogenic niches. As an important component of neurogenic niches, vascular cells interact with NSCs through different signaling mechanisms, which is similar to the characteristics of radiation-induced bystander effect (RIBE). But whether RIBE is involved in neurogenesis inhibition contributed by the damaged vascular cells is unknown. Thus, the purpose of the present study was to investigate the occurrence of RIBEs in non-irradiated bystander NSCs induced by irradiated bEnd.3 vascular endothelial cells in a co-culture system. The results show that compared with the NSCs cultured alone, the properties of NSCs were significantly affected after co-culture with bEnd.3 cells, and further change was induced without obvious oxidative stress and apoptosis when bEnd.3 cells were irradiated, manifesting as a reduction in the proliferation, neurosphere-forming capability and differentiation potential of NSCs. All these results suggest that the damaged vascular endothelial cells may contribute to neurogenesis inhibition via inducing RIBEs in NSCs, thus leading to RIND.

Carnosic acid (CA) is a phenolic diterpene characterized by its high antioxidant activity; it is used in industrial, cosmetic, and nutritional applications. We evaluated the radioprotective capacity of CA on cells directly exposed to X-rays and non-irradiated cells that received signals from X-ray treated cells (radiation induced bystander effect, RIBE). The genoprotective capacity was studied by in vivo and in vitro micronucleus assays. Radioprotective capacity was evaluated by clonogenic cell survival, MTT, apoptosis and intracellular glutathione assays comparing radiosensitive cells (human prostate epithelium, PNT2) with radioresistant cells (murine metastatic melanoma, B16F10). CA was found to exhibit a genoprotective capacity in cells exposed to radiation (p < 0.001) and in RIBE (p < 0.01). In PNT2 cells, considered as normal cells in our study, CA achieved 97% cell survival after exposure to 20 Gy of X-rays, eliminating 67% of radiation-induced cell death (p < 0.001), decreasing apoptosis (p < 0.001), and increasing the GSH/GSSH ratio (p < 0.01). However, the administration of CA to B16F10 cells decreased cell survival by 32%, increased cell death by 200% (p < 0.001) compared to irradiated cells, and increased cell death by 100% (p < 0.001) in RIBE bystander cells (p < 0.01). Furthermore, it increased apoptosis (p < 0.001) and decreased the GSH/GSSG ratio (p < 0.01), expressing a paradoxical radiosensitizing effect in these cells. Knowing the potential mechanisms of action of substances such as CA could help to create new applications that would protect healthy cells and exclusively damage neoplastic cells, thus presenting a new desirable strategy for cancer patients in need of radiotherapy.

Background: Although investigating the probable side effects of post intraoperative radiotherapy wound fluid secretion (PIWFS) is crucial, especially in clinical cases, no report has been published on the effect of PIWFS on the remaining tumor cells (in the vital state) in cavity side margins or surrounding regions. These tumor cells might be directly/indirectly exposed to intraoperative radiation therapy (IORT). Here, for the first time, we investigated the effect of PIWFS on tumor cells of the same patient extracted from the excised tumor in the spheroid form. Methods: We generated 8 human-derived breast tumor spheroids from 4 patient specimens who received to IORT, dissociated and cultured them in microfluidic devices. The spheroids from each sample were treated with the patients’ PIWFS and DMEM medium separately. Two different parameters, called area and number of detached cells (NDCs), were determined and investigated to evaluate the spheroids’ vital and proliferative states. Results: The results showed severe transformation in tumor spheroids’ function into more invasive and proliferative functions after treatment with PIWFS. Conclusion: Although the radiation-induced bystander effect may have a role in this observation, further experiments must be done to better clarify the probable desired or non-desired effects of post-IORT secretion for both the remaining tumor cells and the surrounding immune cells.

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.
Thesis (M.Sc.(Med.Phy.)) -- University of Adelaide, School of Chemistry and Physics, 2013