[en] The extended abstracts which are submitted here present a summary of the proceedings of the 6th International Workshop/12th LH Gray Workshop: Microbeam Probes of Cellular Radiation Response, held at St. Catherine's College, University of Oxford, UK on March, 29th-31st, 2003. In 1993 the 4th LH Gray Workshop entitled ''Microbeam Probes of Cellular Radiation Response'' was held at the Gray Cancer Institute in Northwood. This was organized by Prof BD Michael, Dr M. Folkard and Dr KM Prise and brought together 40 participants interested in developing and applying new microbeam technology to problems in radiation biology (1). The workshop was an undoubted success and has spawned a series of subsequent workshops every two years. In the past, these workshops have been highly successful in bringing together groups interested in developing and applying micro-irradiation techniques to the study of cell and tissue damage by ionizing radiations. Following the first microbeam workshop, there has been a rapid growth in the number of centres developing radiobiology microbeams, or planning to do so and there are currently 15-20 worldwide. Much of the recent research using microbeams has used them to study low-dose effects and ''non-targeted'' responses such bystander effects, genomic instability and adaptive responses. The goal of the 6th workshop was to build on our knowledge of the development of microbeam approaches and the application to radiation biology in the future with the meeting stretching over a 3 day period. Over 80 participants reviewed the current state of radiobiology microbeam research worldwide and reported on new technological developments both in the fields of physics and biology

[en] Highlights: •PERK enhances the sensitivity of cancer cells to ionizing radiation. •Down-regulation of PERK results in enhanced DNA repair. •Ionizing radiation-induced apoptosis is inhibited in PERK-down regulated cancer cells. -- Abstract: Although, ionizing radiation (IR) has been implicated to cause stress in endoplasmic reticulum (ER), how ER stress signaling and major ER stress sensors modulate cellular response to IR is unclear. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) is an ER transmembrane protein which initiates unfolded protein response (UPR) or ER stress signaling when ER homeostasis is disturbed. Here, we report that down-regulation of PERK resulted in increased clonogenic survival, enhanced DNA repair and reduced apoptosis in irradiated cancer cells. Our study demonstrated that PERK has a role in sensitizing cancer cells to IR

[en] For radiobiological applications, the strength of the microirradiation technique lies in its ability to deliver precise doses of radiation to selected individual cells (or sub-cellular targets) in vitro. There is particular interest in studying the risks associated with environmental exposures to α-particle emitting isotopes (which are predominantly due to single-particle effects) and for investigating the so-called 'bystander effect' where non-irradiated cells are seen to respond to signals from nearby irradiated cells. The Gray Cancer Institute charged particle microbeam is one of only two facilities currently in routine use for radiobiology; although a number other facilities are at various stages of development. To be useful in a radiobiological study, a microbeam facility is required to reliably deliver an exact number of particles to a pre-selected sub-cellular target. Furthermore, the low incidence of some biological endpoints means that a large number of cells may have to be individually irradiated (>100,000 cells), therefore some form of automation is essential. Our microbeam uses a 1 μm diameter bore glass capillary to vertically collimate protons, or helium ions accelerated by a 4 MV Van de Graaff. Using ³He²⁺ ions, 99% of cells are targeted with an accuracy of ±2 μm, and with a particle counting accuracy >99%. Using automated cell finding and irradiation procedures, up to 10,000 cells per hour can be individually irradiated

[en] The range of radiobiological experiments requiring microirradiation techniques continues to expand and diversify, creating ever-greater challenges for the designers of such systems. A versatile microbeam for radiation biology must excel in a number of areas. For studies of intracellular cell signalling where it may be of interest to target just the cytoplasm or nuclear membrane, targeting accuracies of micron or less are desirable. Other studies may use endpoints that are rare enough to require the irradiation of hundreds of thousands of cells in order to observe and quantify the effects. Inevitably, this means automating the cell finding, aligning and irradiation steps in order to achieve high cell throughputs. For investigations related to radiation risk, the effect of single particle traversals are paramount, therefore particle counting and single particle delivery are essential. A number of improvements have been implemented to the Gray Cancer Institute charged-particle microbeam, to extend its versatility and to meet these challenges. Specifically, improvements to the speed, alignment accuracy and environmental control have enabled investigations related to cell signalling, low-dose hypersensitivity, genomic instability and the visualisation of DNA repair to be successfully addressed

[en] The Gray Cancer Institute has pioneered the use of X ray focusing techniques to develop systems for micro irradiating individual cells and sub cellular targets in vitro. Cellular micro irradiation is now recognized as a highly versatile technique for understanding how ionizing radiation interacts with living cells and tissues. The strength of the technique lies in its ability to deliver precise doses of radiation to selected individual cells (or sub cellular targets). The application of this technique in the field of radiation biology continues to be of great interest for investigating a number of phenomena currently of concern to the radiobiological community. One important phenomenon is the so called ''bystander effect'' where it is observed that unirradiated cells can also respond to signals transmitted by irradiated neighbors. Clearly, the ability of a microbeam to irradiate just a single cell or selected cells within a population is well suited to studying this effect. Our prototype ''tabletop'' X-ray microprobe was optimized for focusing 278 eV C-K X rays and has been used successfully for a number of years. However, we have sought to develop a new variable energy soft X-ray microprobe capable of delivering focused CK (0.28 keV), Al-K (1.48 keV) and notably, Ti-K (4.5 keV) X rays. Ti-K X rays are capable of penetrating several cell layers and are therefore much better suited to studies involving tissues and multi cellular layers. In our new design, X-rays are generated by the focused electron bombardment of a material whose characteristic-K radiation is required. The source is mounted on a 1.5 x 1.0 meter optical table. Electrons are generated by a custom built gun, designed to operate up to 15 kV. The electrons are focused using a permanent neodymium iron boron magnet assembly. Focusing is achieved by adjusting the accelerating voltage and by fine tuning the target position via a vacuum position feedthrough. To analyze the electron beam properties, a custom built microscope is used to image the focused beam on the target, through a vacuum window. The X-rays are focused by a zone plate optical assembly mounted to the end of a hollow vertical tube that can be precisely positioned above the X ray source. The cell finding and positioning stage comprises an epi-fluorescence microscope and a feedback controlled 3 axis cell positioning stage, also mounted on the optical table. Independent vertical micro positioning of the microscope objective turret allows the focus of the microscope and the X ray focus to coincide in space (i.e. at the point where the cell should be positioned for exposure). The whole microscope stage assembly can be precisely raised or lowered, to cater for large differences in the focal length of the X ray zone plates. The facility is controlled by PC and the software provides full status and control of the source and makes use of a dual-screen for control and display during the automated cell finding and irradiation procedures

[en] Cellular micro-irradiation is now recognised as a powerful technique for understanding how ionising radiation interacts with living cells and tissues. Charged-particle microbeams are uniquely capable of delivering single, or counted multiple particles to selected sub-cellular targets. This capability is particularly useful for studying the risks associated with environmental exposures to α-particle emitting isotopes (such as radon) where exposed cells within the body are unlikely to receive more than one particle traversal. Microbeam methods are also seen as highly appropriate for studying the so-called 'bystander effect' (where unirradiated cells respond to signals transmitted by irradiated neighbours). Using the Gray Laboratory microbeam, we have been able to demonstrate a significant increase in the levels of cell death and DNA damage in a population of cells after irradiating just a few cells within a population. Also, by targeting the cell cytoplasm, we have shown that intra-cellular signalling between the cytoplasm and nucleus can cause DNA damage, showing that direct DNA damage is not required to observe radiation induced effect in cells

[en] Highlights: • Characterization of a compact ²⁴¹Am alpha-source. • Improvement of alpha particle energy spectrum with a 3D printed collimator. • Proven feasibility of a low-cost alpha-irradiation setup for in vitro assays. • Alpha particle RBE for 10% survival in PC-3 is 3.66. A compact in-house alpha particle source has been developed and fully characterized. The irradiation source is a large area, 25 cm², 5.4 MeV average energy ²⁴¹Am source, above which a Mylar dish containing a monolayer of target cells can be placed at defined positions. The source uniformity, flux, particle energy and dose rate were determined experimentally. The dose rate to the nucleus at the closest position was 1.57 Gy/min. Furthermore, a 3D printed collimator was tested and found to improve the uniformity of the energy spectra of particles reaching the target. For validation, prostate PC-3 cells were irradiated in our experimental setup with absorbed doses up to 2 Gy and for reference compared with cells irradiated with conventional X-rays with doses up to 8 Gy. The Relative Biological Effectiveness for alpha particles at 10% survival was 3.66$\pm$ 0.40 agreeing with previously published data. Data presented here show the feasibility of utilising a low-cost alpha-irradiation source for accurate in vitro assays to better understand the radiobiological effects of high LET alpha particles.

[en] Highlights: ► KNK437, a benzylidene lactam compound, is a novel radiosensitizer. ► KNK437 inhibits AKT signaling and abrogates the accumulation of HIF-1α under hypoxia. ► KNK437 abrogates hypoxia induced resistance to radiation. -- Abstract: KNK437 is a benzylidene lactam compound known to inhibit stress-induced synthesis of heat shock proteins (HSPs). HSPs promote radioresistance and play a major role in stabilizing hypoxia inducible factor-1α (HIF-1α). HIF-1α is widely responsible for tumor resistance to radiation under hypoxic conditions. We hypothesized that KNK437 sensitizes cancer cells to radiation and overrides hypoxia-induced radioresistance via destabilizing HIF-1α. Treatment of human cancer cells MDA-MB-231 and T98G with KNK437 sensitized them to ionizing radiation (IR). Surprisingly, IR did not induce HSPs in these cell lines. As hypothesized, KNK437 abrogated the accumulation of HIF-1α in hypoxic cells. However, there was no induction of HSPs under hypoxic conditions. Moreover, the proteosome inhibitor MG132 did not restore HIF-1α levels in KNK437-treated cells. This suggested that the absence of HIF-1α in hypoxic cells was not due to the enhanced protein degradation. HIF-1α is mainly regulated at the level of post-transcription and AKT is known to modulate the translation of HIF-1α mRNA. Interestingly, pre-treatment of cells with KNK437 inhibited AKT signaling. Furthermore, down regulation of AKT by siRNA abrogated HIF-1α levels under hypoxia. Interestingly, KNK437 reduced cell survival in hypoxic conditions and inhibited hypoxia-induced resistance to radiation. Taken together, these data suggest that KNK437 is an effective radiosensitizer that targets multiple pro-survival stress response pathways.

[en] Radiotherapy is commonly planned on the basis of physical dose received by the tumour and surrounding normal tissue, with margins added to address the possibility of geometric miss. However, recent experimental evidence suggests that intercellular signalling results in a given cell’s survival also depending on the dose received by neighbouring cells. A model of radiation-induced cell killing and signalling was used to analyse how this effect depends on dose and margin choices. Effective Uniform Doses were calculated for model tumours in both idealised cases with no delivery uncertainty and more realistic cases incorporating geometric uncertainty. In highly conformal irradiation, a lack of signalling from outside the target leads to reduced target cell killing, equivalent to under-dosing by up to 10% compared to large uniform fields. This effect is significantly reduced when higher doses per fraction are considered, both increasing the level of cell killing and reducing margin sensitivity. These effects may limit the achievable biological precision of techniques such as stereotactic radiotherapy even in the absence of geometric uncertainties, although it is predicted that larger fraction sizes reduce the relative contribution of cell signalling driven effects. These observations may contribute to understanding the efficacy of hypo-fractionated radiotherapy. (paper)

[en] Variations in proton relative biological effectiveness (RBE) with linear energy transfer (LET) remain one of the largest sources of uncertainty in proton radiotherapy. This work seeks to identify metrics which can be applied to mitigate these effects in treatment optimisation, and quantify their effectiveness. Three different metrics—dose, dose  ×  LET and an LET-weighted dose defined as where is the dose-averaged LET—were compared with in vitro experimental studies of proton RBE and clinical treatment plans incorporating RBE models. In each system the biological effects of protons were plotted against these metrics to quantify the degree of variation introduced by unaccounted-for RBE uncertainties. As expected, the LET-dependence of RBE introduces significant variability in the biological effects of protons when plotted against dose alone. Plotting biological effects against dose  ×  LET significantly over-estimated the impact of LET on cell survival, and typically produced even larger spreads in biological effect. LET-weighted dose was shown to have superior correlation to biological effect in both experimental data and clinical plans. For prostate and medulloblastoma treatment plans, the average RBE-associated variability in biological effect is  ±5% of the prescribed dose, but is reduced to less than 1% using LET-weighting. While not a replacement for full RBE models, simplified metrics such as this LET-weighted dose can be used to account for the majority of variability which arises from the LET-dependence of RBE with reduced need for biological parameterisation. These metrics may be used to identify regions in normal tissues which may see unexpectedly high effects due to end-of-range elevations of RBE, or as part of a more general tool for biological optimisation in proton therapy. (paper)