The objective of the work described in this manuscript was to develop a novel assay to measure direct and bystander radiation damage on an in vitro
3D human skin tissue model. The use of a biological model that closely resembles normal human tissue offers great potential for the investigation of non-targeted radiation effects in a more relevant environment, where cell signalling and direct cell-cell contact play a critical role. Although 3D models have already been used for similar studies, so far it has not been possible to investigate the bystander contribution for induction of critical DNA damage events. This was due to difficulty in performing single cell assays in tissues, while preserving their spatial correlation. We have used the described method to investigate the induction of micronuclei following partial irradiation (50 μm wide line across the tissue diameter) with 3.5 MeV protons in order to compare the results with available published data and demonstrate the feasibility of the protocol. The micronuclei assay has been chosen as there are extensive literature data (Belyakov et al., 2003
, Prise et al., 1998
) confirming the validity of an assay as the measure for bystander effect and offering suitable data for comparison in 2D systems (explants and isolated cell systems). The reproducibility of the data and the low level of damage detected in the control samples seem to support the use of such approach for the investigation of other biological endpoints.
The developed slicing and harvesting protocol has been shown to be able to recover a very large fraction of the tissue cells obtaining a still viable single cell solution. The number of cells recovered was in agreement with the number calculated with a simple geometrical approach and with the estimation provided by the tissue supply company (MatTek). The purpose built microtome to slice live tissues has proved to be very precise and effective, easy to use and economically affordable. Three different Cytochalasin-B concentrations have been tested for an incubation time up to 72 h to determine the cell division rate and the best conditions for micronuclei scoring. The maximum number of binucleated cells was achieved for a 3 μg/ml concentration and 48 h incubation with no evident improvement for longer incubations or higher concentrations. Although the fraction of binucleated cells measured (~60 %) was lower than that observed in the same in vitro cell cultures (this may be due to the longer trypsin treatment), it is still adequate to perform the proposed studies and the technique demonstrates that an overall viable single cell population was produced. Additionally, the background frequency of micronuclei scored in the control samples is considerably low (0.72 ± 0.37 % of micronucleated cells) and very reproducible, making the model suitable for straightforward statistical analysis. The lack of increase in the micronuclei frequency in control samples which had been sliced compared to that measured in intact tissues, also indicates that no considerable stress or alteration is introduced by the slicing process.
Partial irradiation of the samples resulted in an elevated micronuclei frequency not only in the stripes containing the direct irradiated cells but also in some of the adjacent stripes with unexposed cells. The damage detected in those cells cannot be attributed to the effect of proton scattering (< ±15 μm as from TRIM simulations) or secondary electrons (max energy ~7.5 keV, range <2.5 μm). The magnitude of the bystander effect observed seems to be dose independent and clearly detectable in our 3D tissue models for doses as low as 0.1 Gy. Furthermore, the data indicate a higher level of micronuclei in the central portion of the sample (i.e. closer to the irradiation site) with evidence of a decreasing but still detectable effect towards the edges. This suggests a range for the bystander response of several millimetres, with potential critical consequences for radiotherapy and radioprotection as it essentially increases the volume and the number of cells affected. The results are in agreement with previous data (Belyakov et al., 2005
) obtained irradiating the same biological system with a precise number of α-particles (microbeam irradiation) and scoring for micronuclei formation in fixed sliced sections of the tissue. The longer range of the damage reported in this manuscript (elevation of micronuclei detected up to a few millimetres from the irradiated area) could be accounted by the different irradiation exposures. While Belyakov and colleagues irradiated about 80 locations along the diameter of the sample (10 α-particles every 100 μm), our samples experienced a more uniform exposure (all cells along the 50 μm wide line across the sample diameter were irradiated). Considering also the higher penetration of 3.5 MeV protons (190 μm compared to 60 μm of 7.2 MeV α-particles), this implies a greater number of cells directly irradiated. The longer range of the damage measured could therefore suggest a link between the number of cells targeted and the strength of the bystander signal. On the other hand, the higher fraction of micronuclei detected in the central area of the sample and the long range of the effect, support the hypothesis that signal(s) are generated by the cells directly damaged by radiation and propagate cell-by-cell (whether they are damaged or not) with great efficiency. Due to the overall low level of damage induced (<3 % micronucleated cells), if the bystander signal were to be propagated only by the damaged cells (although with high efficiency), it would have been reasonable to expect a rapid decrease with distance from the irradiation site. The long range of the effect seems to suggest that cells that do not exhibit damage are also involved in the signal propagation.
Interestingly, almost no significant increase above the background level was measured for the 0.1 Gy - 0.5 mm stripes dose point. A possible explanation could lie in the different number of cells scored (~1000 samples for the narrow stripes against more than 2000 for all other cases) as forced by the lower number of cells recovered. However under the same experimental conditions (0.5 mm wide stripes), a significant bystander effect was detected for the 1 Gy dose point with the 0.5 Gy relative data showing an ambiguous response. Further investigations are required by expanding the dose range and/or by slicing the samples into finer stripes.
In conclusion, the excellent control levels, low variability and trend agreement with other bystander measurements in tissue systems indicate that the approach described in this manuscript is suitable for accurate bystander investigations in complex 3D samples. Compared to the traditional technique where the sample is left intact and then fixed/sectioned to score in situ damage, this method offers the flexibility to live section the samples at any time post irradiation (while preserving cell activities and proliferation for longer periods) to specifically address questions related to the transmission of extracellular signals. Moreover, dissociation of the samples and further culturing of single cells widen the range of biological end points while at the same time preserving same spatial information.