MRTN-CT-2003-503923     CELLION
Studies on cellular response to targeted single ions using nanotechnology

Project overview
Estimation of radiation hazards requires profound knowledge on interaction of ions with biological cells and tissue. The action of exposure to very low dose radiation is presently only estimated by high dose extrapolation. If low dose effects are studied by irradiating a cell culture by a conventional broad ion beam, only some few cells are hit. As in a control culture some cells always die spontaneously, the result is of low statistical value. Therefore, a better aiming accuracy of bombarding ions is needed to choose exactly which cells and which organelles within were damaged by traversing ions. To solve the problem a network of high accuracy ion microprobes (collimated or focused) was created. Consequences of radiation damage and pathways of damage repair will be studied using various physical and molecular biology methods.

Overall Objectives
The research objectives to be reached in the project are divided into two main groups: biology and instrumentation, each group with its own co-ordinator.

I. Research objectives in biology:
Single-hit studies with sub-cellular resolution will provide insights into a number of questions concerning cell damage and repair. It will be found how the radiation sensitivity varies in different regions of the cell and what information does this provide about its structure. It will answer the question whether the observed patterns of radiation sensitivity depend on the chromatin distribution, or on the induced DNA damage, or on the localization of DNA repair capacity sites, or reflect long range interaction between damage sites. The observation of neighbouring non-irradiated cells will clarify whether the damage can be transferred from hit to non-hit cells (bystander effect). The answers to the above questions are of relevance to estimate the cancer risk due to natural radioactivity in the environment and to cosmic radiation and the occupational health hazards of workers with radioactive materials used in industry or medicine. It will help also to optimize the methods of radiotherapy. The answers to the above questions are of relevance to estimate the cancer risk due to natural radioactivity in the environment and to cosmic radiation and the occupational health hazards of workers with radioactive materials used in industry or medicine. It will help also to optimize the methods of radiotherapy.
The following investigations will be performed:

Morphological mapping of radiation response and DNA damage induction
A relation between the induction of lethal lesions and the cell morphology will be established. A distribution of radiation induced DNA damage across the cell will be determined and the importance of the nuclear and chromosome aberrations will be checked. The investigation is running already and a start of the network will better coordinate its progress. Any progress in the tasks listed in the Table will improve accuracy and quality of the results.

p53 expression and apoptosis; gene induction studies.
In the project the role of nuclear versus non-nuclear targeting of radiation to the induction of p53 and the cell cycle delay will be studied. A relation between nuclear and non-nuclear targeting and the triggering of apoptosis will be established. By targeted ionizing radiation to sub-cellular locations it will be established the role of radiation induced signal transduction pathways in processes such as proliferation, apoptosis, stress factor, genomic instability and induced radioresistance. These studies may provide fundamental information required for development of cancer therapies involving the manipulation of cell signalling pathways. The studies are already undertaken by several groups and especially participants 2, 3 and 4 are advanced in the investigation.

Cell-to-cell communication
Different signalling pathways leading to the bystander effect and the influence of the effect to predictions of low dose radiation induced hazards will be studied. An intelligent wet cell chamber will be used to measure in situ a time and space variation of the Ca++ signal with a sub-cellular resolution. A success in the task will result in a new, important tool changing the quality of the investigation.

Cell cycle modelling
Mathematical models of the response of cells and tissues to ionising radiation will be refined (this is a task #5 listed in the Table I). The models will be used to predict the outcome of radiotherapy treatment strategies and the influence of factors such as such as radiation quality, hypoxic status, hypersensitivity, non-targeted effects (such as the bystander effect) and radio-resistance through the cell cycle. Data from the microbeam studies will underpin the development of the model, which in turn, will drive aspects of the experimental research. The goal is to facilitate the development of new and fully optimized radiotherapy fractionation schedules for clinical practice.

II. Research objectives in instrumentation:
Single ion hit facilities will be developed at the ion microprobes with parameters discussed below:

Focusing versus collimation of the microprobe ion beams
Until now, microprobes equipped with a single ion hit facility use beams collimated by a pinhole or a capillary. The majority of other groups developing single ion hit facilities, including most of the participants of the present project, use focused beams. It is expected that with focused beams a better targeting accuracy can be reached. Using scanning focused beams the number of cells that can be irradiated in a given time will be considerably increased. The rapid scanning will make feasible to study routinely biological endpoints that occur infrequently, such as cell transformation (an important endpoint with regard to cancer induction by radiation). It will also enable investigation of larger number of cell lines and will facilitate studies on synchronised cell phases where rapid scanning is vital as a phase can be complete in ~1 hour.

Single ion detection
Very high detection efficiency (close to 100%) for single ions is required in the project with simultaneous condition that the detector thickness cannot considerably change the initial energy of ions. In order to achieve that detectors made of thin diamond foil doped with boron, of silicone nitride foil covered with Au + CsI and o f very thin scintillator will be developed and checked for ions with different energy and different value of Z.

Fast beam switch and target holder for irradiations of wet cells
The reduction of the beam intensity and a fast beam switch preventing hits of the cell by the next ion were already solved by the participants of the project. Individual solutions within the network will be compared and in case of need exchanged for better ones. Several designs of wet cell chambers were already successfully examined. However, the participants of the project wish to develop an intelligent wet cell chamber enabling quantitative measurements of the cell signals in situ during irradiations. This will be done by construction of adhesion points in the Petri dish and an assembly of nanowires with a facility to measure ultra low currents.

Microscopy, cell recognition and positioning
Many experiments planned with single-ion-hit microprobes require irradiation of hundreds or thousands of cells with precisely the same localization of the ion traversal through the cell. The experiment must be finished quickly, because the cells will not survive sufficiently long outside the incubator. After the irradiation, the dish with irradiated cells will be transferred into the stage of a microscope and the positions of particular cells should be recognized again. This requires an automatic system of cell recognition and positioning. Until now only stained cells could be recognized by computer controlled systems without significant amount of mistakes. However, the staining procedure introduces an unnecessary disturbance to the functions of the cell. Three ways were chosen to reduce this disturbance: (a) to reduce the amount of staining substances, (b) to develop a new optical system and computer program of the cell recognition based on the phase analysis of the microscopy image and (c) what will ease the position recognition.

Ion Beam Analysis
High resolution ion beam analysis on in vitro living cells and tissue will be undertaken. This will allow parallel studies of molecular changes within the cell and at the cell surface, for example shifts in intra-cellular trace element distribution or changes in protein or drug uptake. These studies will produce important insights into the influence of 'bystander' effects on the outcome of radiation in tumours and normal tissues. They will also be unique in their ability to quantify risk from low level radiation exposure to individual cells within a defined population.