J. Radiat. Res., 53, 395–403 (2012)
Radiobiological Effectiveness of Laser Accelerated Electrons in
Comparison to Electron Beams from a Conventional
Linear Accelerator
Lydia LASCHINSKY1*, Michael BAUMANN1, Elke BEYREUTHER2, Wolfgang ENGHARDT1,2,
Malte KALUZA3, Leonhard KARSCH1, Elisabeth LESSMANN2, Doreen NAUMBURGER1,
Maria NICOLAI3, Christian RICHTER1,2, Roland SAUERBREY2,
Hans-Peter SCHLENVOIGT3 and Jörg PAWELKE1,2
Laser particle acceleration/Laser radiotherapy/Cell response to electrons/Pulsed irradiation.
The notable progress in laser particle acceleration technology promises potential medical application
in cancer therapy through compact and cost effective laser devices that are suitable for already existing
clinics. Previously, consequences on the radiobiological response by laser driven particle beams characterised by an ultra high peak dose rate have to be investigated. Therefore, tumour and non-malignant cells
were irradiated with pulsed laser accelerated electrons at the JETI facility for the comparison with continuous electrons of a conventional therapy LINAC. Dose response curves were measured for the biological
endpoints clonogenic survival and residual DNA double strand breaks. The overall results show no significant differences in radiobiological response for in vitro cell experiments between laser accelerated pulsed
and clinical used electron beams. These first systematic in vitro cell response studies with precise dosimetry to laser driven electron beams represent a first step toward the long term aim of the application of
laser accelerated particles in radiotherapy.
INTRODUCTION
Until now ion radiotherapy including protons and heavier
particles is limited to a few large scale facilities, e.g. HIT or
NIRS, due to the requirement of a synchrotron or cyclotron
accelerator often associated with large gantry systems.1,2)
The development of high intensity laser particle acceleration
over recent years has been discussed as a potential alternative acceleration technology for ion radiotherapy.3–8)
The laser particle acceleration (cf. Fig. 1) was firstly proposed theoretically for electrons9) and has rapidly progressed
to the generation of intense electron10–13) and ion pulses.14–16)
Several studies have been published demonstrating simulations and proposals of laser particle acceleration improve*Corresponding author: Phone: +49 351 458 7436,
Fax: +49 351 458 7311,
E-mail: [email protected]
1
OncoRay – National Center for Radiation Research in Oncology, Medical
Faculty Carl Gustav Carus, Technische Universität, Fetscherstraße 74,
01307 Dresden, Germany; 2Helmholtz-Zentrum Dresden-Rossendorf,
Bautzner Landstraße 400, 01328 Dresden, Germany; 3Institute of Optics and
Quantum Electronics, Friedrich-Schiller-University, Max-Wien-Platz 1,
07743 Jena, Germany.
doi:10.1269/jrr.11080
ments as well as the essential beam transport for the therapeutic application.3,17–23) As consequence the laser plasma
community is promising compact and potentially very cost
effective laser facilities4–7,24–27) that are suitable for already
existing clinics. Among others the specific property of laser
accelerated particle beams is the ultra high pulse dose rate
(≥ 109 Gy/sec) exceeding those of conventional accelerated
particle beams by several orders of magnitude.28–31) Before
clinical application several strict requirements with respect
to beam parameters like dose rate, particle energy, stability,
quality assurance and patient safety need to be fulfilled.5,6,27,32) Moreover, the physical and real-time dosimetric characterization and the investigation of radiobiological
consequences of novel laser accelerated particle beams are
essential. These imply complex translational investigations
starting from in vitro cell irradiation to animal experiments
and finally clinical trials with patients.
Even though the development of laser driven proton and
ion therapy is the long term goal, the first laser accelerated
particles being experimentally available with sufficient high
energy and intensity as well as high pulse frequency relevant
for radiotherapeutic application are laser accelerated electrons. Accordingly, laser accelerated electrons produced at
the JETI laser facility in Jena were used for the experiments
presented here. Beyreuther et al.28) have shown the general
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L. Laschinsky et al.
applicability of the JETI system for in vitro cell experimentation. The aim of the present study was to determine dose
response curves and to compare the radiobiological effectiveness of laser generated electron pulses with that of a
continuous beam delivered by a clinical LINAC for both
non-malignant and tumour cells.
MATERIALS AND METHODS
Cell lines and cell culture
The undifferentiated human squamous cell carcinoma
FaDu,33,34) established as subline of the original FaDu cell
line,35) was maintained in Dulbecco’s minimum essential
medium with 4.5 g/l stable glutamin (Biochrom, Berlin,
Germany) containing 10% FBS (Sigma-Aldrich, Munich,
Germany), 1 mM sodium pyruvate, 20 mM HEPES, 1%
100x non-essential amino acids (all from PAA, Pasching,
Austria) and 1% Penicillin/Streptomycin (Biochrom). The
non-malignant, immortalised, normal mammary gland epithelium cell line 184A1 (CRL-8798, ATCC, LGC Promochem,
Wesel, Germany), originally established from a mammoplasty,36) was grown in serum-free mammary epithelial basal
medium supplemented with MEGM SingleQuots® (both
from Lonza, Cologne, Germany), 50 mg/ml prostaglandin E1
(Calbiochem, Darmstadt, Germany) and 5 μg/ml human apotransferrin (Sigma-Aldrich). Both cell cultures were maintained at 37°C, 5% CO2, 95% humidification and routinely
checked for mycoplasma using Venor®GeM (Biochrom).
Exponentially growing cells were seeded two days before
irradiation in 8-well-chamber-slides (PermanoxTM, Nunc,
Wiesbaden, Germany) for γH2AX/53BP1 assay at a density
of 2.5 × 104 cells/cm2 and for clonogenic assay in 35 mm
petri dishes (Greiner bio one, Frickenhausen, Germany) at a
density of 1.0 × 105 cells/cm2. One day before irradiation,
the cell culture medium was completely exchanged. Immediately before irradiation the cell samples were completely
filled up with culture medium and finally closed with sterile
Parafilm® (24 h in 80% Ethanol, at least 2 h UV light). The
cell vitality and plating efficiency under these conditions was
constant over the tested period up to 4 hours. Unirradiated
cell samples were prepared in the same way like the irradiated probes and were used as controls.
Irradiation with laser accelerated electrons at JETI
Customation of the Jena Titanium:Sapphire laser system
(JETI) for irradiation of cells was previously described.28)
The further optimized setup used for the experiments reported here is shown in Fig. 2. Two basic improvements were
introduced: a more effective filtering of low energetic electrons by a permanent S-bending magnetic energy filter with
0.12 T and the control of the electron energy distribution.
The energy spectrum was controlled directly with an electromagnetic spectrometer detecting the intensities of the
deflected electrons with Gafchromic™ EBT-1 radiochromic
films (ISP Corp., Wayne, USA) or imaging plates (Fujifilm,
Tokio, Japan). Additionally, the spectrum was reconstructed
on basis of depth dose distributions that were measured on
a daily basis with EBT-1 film stacks. The obtained energy
spectra were in good agreement to each other, covering an
energy range of 3–20 MeV and showing a maximum of the
energy spectrum at approximately 6.5 MeV.37)
Online monitoring and irradiation control were performed
with a Roos ionization chamber type 34001 (PTW, Freiburg,
Germany) and a Faraday cup (manufactured in house) both
providing real-time dose information. Additionally, EBT-1
films were placed in front of each cell sample to determine
retrospectively the absolute dose delivered to the cells. In
order to correct the dose differences between the film location in front of the cell culture vessel and at the cell monolayer location, a mean dose correction factor of 1.064 was
determined by averaging the dose ratios obtained on a daily
basis for the two vessel types with films at both positions.
At this, water equivalent material RW3 (PTW) was placed
directly behind the film at cell position to account for backscattered electrons from the cell media.
For cell irradiation each sample was placed in a PMMA
sample holder and was set with the vessel bottom facing to
the horizontal beam coming from direction of the vacuum
window minimizing the material in front of the cell monolayer (cf. Fig. 2). Doses in the range of 0.4 Gy to 10.2 Gy
were delivered to the cells by 125 to 4900 electron pulses
with a repetition rate of 2.5 Hz. A peak dose rate of 2.4 ×
109 Gy/sec was supposed by the estimated pulse duration of
1 × 10–12 sec based on laser pulse length, acceleration process and electron beam transport. In spite of the ultra high
peak dose rate a rather low mean dose rate of (0.361 ±
0.013) Gy/min while irradiation time, averaged over all 163
cell irradiation, was determined. This was a substantial
improvement compared to the 0.24 Gy/min achieved previously.28) Nevertheless the practically obtained still low mean
dose rate is the result of the limit with the current laser system and of the complex optimization of the necessary beam
parameters for cell irradiation like dose homogeneity, beam
stability, reproducibility, intensity and spot size.
Irradiation using a clinical electron linear accelerator
A clinical electron linear accelerator (LINAC, Oncor
Impression, Siemens AG, Healthcare, Erlangen, Germany)
located in Dresden was used for in vitro cell irradiation. The
LINAC was calibrated using the TRS-398 dosimetry protocol.38) The electron beam of the clinical setting was delivered
with a dose rate of 3 Gy/min, whereas the energy of 6 MeV
was chosen in accordance to the maximum of the energy
spectrum generated by the JETI laser accelerator facility. It
has to be noted that the LINAC electron beam is also a
pulsed beam. The beam was delivered with pulse duration of
4 μsec at a pulse frequency of 50 Hz which results in a duty
cycle of 2 × 10–4. In comparison to the duty cycle of 2.5 ×
In Vitro Cell Response to Laser Electrons
10–12 of the laser electron beam, the LINAC electron beam
is considered in the following text as continuous beam.
Both vessel types were irradiated in horizontal position
with the beam coming from above entering the cell monolayer through the top of the petri dish or the bottom of the
8-well-chamber-slide. In order to position the cell monolayer
at the reference depth, the vessels were covered by PMMA
slabs of 10 mm and 2 mm for the 8-well-chamber-slides and
petri dishes, respectively. To ensure charged particle equilibrium in the whole vessel, the vessel was surrounded by RW3
(PTW).
397
performed at the JETI laser facility in Jena and at the clinical
therapy LINAC in Dresden. In order to investigate possible
diversities of cell cultivation and distinct radiosensitivity Xray reference irradiation has been performed in parallel to
each electron experiment. Dose response curves were determined for each biological endpoint at the two 200 kVp X-ray
tubes – Maxishot 200 Y.TU/320-D03 (Yxlon International
A/S, Taastrup, Denmark) in Jena and Isovolt 320/20
(Röntgen Seifert, Ahrensburg, Germany) in Dresden – with
similar radiation quality, respectively. Characteristics of both
tubes and absolute dosimetry under consideration of the different irradiation geometries were previously described.39,40)
X-ray reference irradiation
Two independent electron irradiation experiments were
Fig. 1. High intensity laser acceleration of electrons. The laser
beam from the Jena Titanium:Sapphire (JETI) terawatt laser system
is focused inside a vacuum chamber by a parabola (right) into a
helium gas jet (centre). Laser light intensities of about 1018 W/cm2
are achieved for femtoseconds pulse length, accelerating electrons
in distances of a few 100 micrometers to MeV energy to the left
(Photograph: Research group Ultraphotonics-Polaris, Institute of
Optics and Quantumelectronics, Friedrich-Schiller-University
Jena, Germany).
Fig. 2. Experimental setup of the laser accelerator JETI: Laser
pulses were focused into a helium gas jet. During the interaction,
the laser pulse generates plasma and accelerates electrons in forward direction. The electrons were energetically filtered by a pair
of permanent magnets and exited the vacuum system through a
1 mm thin aluminium window. Behind this filter arrangement, the
electron beam propagates in air and passes the cell monolayer sample (diameter of 35 mm). For relative online dosimetry an ionization chamber and a Faraday cup were used. Radiochromic films
allowed an absolute dosimetric determination directly at cell position. The spectrometer, image plates and radiochromic films were
used to determine the energy spectrum.
Fig. 3. Dose response curves of clonogenic survival after irradiation of normal tissue cell line 184A1 (left) as well as tumour
cell line FaDu (right) with pulsed laser generated electron beam (red curves) and continuous electron beam delivered by a
therapeutic LINAC (blue curves). All curves are presented in consideration of their 95% confidence intervals.
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L. Laschinsky et al.
Thus, by a focus-to-object distance of 464 mm a dose rate
of 1.39 Gy/min for the Yxlon tube and a dose rate of
1.21 Gy/min at a focus-to-object distance of 460 mm for the
Isovolt tube were determined, respectively. Dose homogeneity over the irradiated field was controlled with EBT-1
films.41) At both X-ray tubes, the cell samples were placed
on a horizontal PMMA holder and were irradiated at room
temperature from above.
The X-ray reference irradiation performed in Dresden and
Jena were used in order to consider different cell conditions
at the two experimental sites by introducing a scaling factor.
This includes all factors (e.g. from laboratory equipment
such as incubator or reported in42)) which influence the dose
response curve but could not be reflected by the subtraction
of the background level of the unirradiated control samples.
The scaling factor was calculated by the ratio between the
slopes of the two X-ray dose response curves, i.e. the curve
determined in Dresden and Jena, respectively. For cell survival, both cell lines showed no significant difference
between the X-ray dose response curves under consideration
of their 95% confidence interval and correspondingly the
scaling factor was 1. The scaling factor was used to adapt
the LINAC (Dresden) electron dose response curve for comparison with the JETI (Jena) electron dose response curve.
The resulting data were fitted in a multilevel fit as described
below. Uncertainties by using the scaling factors have been
considered by error propagation.
In order to quantify the effect due to low mean dose rate of
laser electrons a low dose rate X-ray irradiation (0.35 Gy/min,
5 mA, LDR) was performed in addition to the usually utilized dose rate (1.39 Gy/min, 20 mA, HDR).
Colony forming assay
Cell survival was determined by standard colony forming
assay. Immediately after irradiation the cells were rinsed,
trypsinised and seeded as duplicates for each dose point in
6 different concentrations using 6-well-plates (Greiner bio
one). The cells were incubated for 13 days (37°C, 5% CO2,
95% humidity), fixed for 15 min with ice cold 80% ethanol
and stained for 10 min with crystal violet (Clin-Tec, Munich,
Germany). Colonies of more than 50 cells were considered
as survivors.
Immunohistochemistry
Residual DNA double strand breaks (DSB) were detected
immunohistochemically by the γH2AX/53BP1 technique, as
previously described.34,40) Irradiated cells were incubated
(24 h, 37°C, 5% CO2, 95% humidity) and accordingly fixed
in natural buffered 1% formalin (15 min), washed once with
PBS/Glycine (Merck, Darmstadt, Germany) and permeabilised with ice cold triton X-100 solution (5 min, 3x, 0.01%
in PBS, BDH Biochemicals, Dorset, England). Subsequently, the cells were washed in PBGT solution consisting of
0.5% gelatine (BDH Prolabo®) and 0.025% TWEEN® 20
(Sigma-Aldrich) in PBS and sequently stained with antiphospho-Histone H2AX (1:1000 in PBGT, 1 h, upstate by
millipore, Schwalbach, Germany), Alexa Fluor® 594 goat
anti-mouse IgG (1:400 in PBGT, 30 min, Invitrogen,
Karlsruhe, Germany), anti-53BP1 (1:3000 in PBGT, 1 h,
Novus Biologicals, Littleton, USA) and Alexa Fluor® 488
goat anti-rabbit IgG (1:1000 in PBGT, 30 min, Invitrogen).
The incubation was performed at 37°C in a wet chamber.
After each staining step the cells were washed with PBGT
(5 min, 3x). Finally, the cell samples were mounted with
DAPI/Vectashield mounting medium (Vector Laboratories,
Burlingame, USA) and capped with a coverslip. The
γH2AX/53BP1 foci were counted visually under 1000-fold
magnification using an Axiovert S100 fluorescence microscope (Carl Zeiss, Jena, Germany) and the HC TriplebandFilterset DAPI/FITC/TxRED (AHF Analysentechnik,
Tübingen, Germany). For each dose point 100 randomly
chosen intact nuclei, which was controlled by DAPI staining, were analysed.
Statistical Analysis
The dose dependent surviving fractions (SF) were determined for two to three independent experiments for all radiation qualities and cell lines. For this, the linear-quadratic
model SF(D) = exp(–αD – βD2) was applied to fit the data,
with SF as the surviving fraction in dependence on dose D
and α and β as fitting parameters. The resulting number of
γH2AX/53BP1 foci was gained from at least three independent experiments for each dose point, cell line and all beam
qualities. For every data point the mean background level of
γH2AX/53BP1 foci, as obtained by the several control samples for each cell line and radiation quality, was subtracted.
The resulting data points were fitted according to the linear
model F(D) = αD where F represents the value of mean foci
per cell in dependence on dose D and α the fit parameter.
All dose response curves were fitted by the software Origin
8 (OriginLab Corporation, Northampton, USA) in a multilevel fit considering both the uncertainties in dose and in the
corresponding biological endpoints.
RESULTS
Figure 3 compares the cell survival curves achieved by
irradiating the two cell lines (184A1, FaDu) with laser accelerated electrons of ultra high peak dose rate but low mean
dose rate and continuous, clinical used LINAC electrons.
The associated survival dose response curves to the 200 kVp
X-ray reference irradiation at each experimental site were
consistent for both cell lines allowing the direct comparison
of the two electron dose response curves without applying a
scaling factor. No significant difference in absolute cell survival level was determined between the radiobiological dose
responses to both electron beams. But the shape of the two
survival curves caused by both electron beam qualities dif-
In Vitro Cell Response to Laser Electrons
fers slightly.
The variation in the shape of survival curves caused by the
low mean dose rate of JETI laser electrons affirm the well
known dose rate effect. A clear modification in the shape of
the survival curve was determined for the irradiation of
184A1 with JETI electrons. The low mean dose rate reduced
the shoulder of the survival curve as seen for the red line and
became more similar to a straight line than the blue one on
the log-linear diagram. With the FaDu cells this effect was
less obvious. For affirmation of the observed mean dose rate
effect in vitro cell experiments were performed with
200 kVp X-ray reference irradiation using the low dose rate,
equal to JETI electrons, in comparison to the conventional
dose rate of clinical used LINAC electrons. Figure 4 presents
the survival curves determined for both dose rate regimes for
184A1 and FaDu. All over again in both cell lines as expected the grey lines, low dose rate, showed a less expressed
shoulder and are therefore more similar to a straight line
Table 1. Coefficients and standard error (SE) of the linearquadratic model for the survival curves presented in Fig. 3 und
Fig. 4 as well as the coefficients of determination R2 of the
curves.
Cell line Radiation quality
FaDu
β (Gy–2)
than the black ones representing the usually used dose rate.
The coefficients derived from the fitting procedure are presented in Table 1 together with their standard errors and
coefficients of determination R2.
For residual DNA DSB 24 h after irradiation, as second
radiobiological endpoint, a difference in dose response
curves was obtained for both cell lines by analysing the Xray reference irradiation data in Jena and Dresden. The
investigation of residual DNA DSB was more sensitive e.g.
Table 2. The slopes of the residual DNA DSB linear regression data before and after scaling were given for the 200 kV
X-ray reference irradiation and for the electron experiments
of both cell lines, respectively.
Cell line Dose response curves
Slope
before scaling
Slope
after scaling
FaDu
X-ray Jena
1,012 ± 0,057
–
X-ray Dresden
1,299 ± 0,053 1,010 ± 0,042
JETI Jena
0,903 ± 0,027
LINAC Dresden
1,288 ± 0,048 0,999 ± 0,038
X-ray Jena
0,858 ± 0,045
X-ray Dresden
0,874 ± 0,043 0,858 ± 0,042
JETI Jena
0,500 ± 0,046
LINAC Dresden
0,851 ± 0,035 0,835 ± 0,038
184A1
R2
0,259 ± 0,039 0,037 ± 0,008 0,9899
JETI
0,244 ± 0,039 0,043 ± 0,007 0,9414
LINAC
1
0,316 ± 0,033 0,062 ± 0,006 0,9466
1
X-ray, LDR
0,548 ± 0,037 0,026 ± 0,007 0,9151
JETI
0,416 ± 0,047 0,004 ± 0,006 0,9444
Cell line
LINAC
0,241 ± 0,045 0,030 ± 0,008 0,9589
FaDu
X-ray, HDR2
0,245 ± 0,058 0,049 ± 0,010 0,8469
2
0,424 ± 0,035 0,028 ± 0,006 0,9053
X-ray, HDR
184A1
α (Gy–1)
X-ray, LDR
1
2
Experiments performed in Jena and in Dresden.
399
–
–
–
Table 3. Mean number of background γH2AX/53BP1
foci for both cell lines and the two experimental locations.
184A1
Experiment location
Mean background level
Jena
0,376 ± 0,023
Dresden
0,492 ± 0,039
Jena
0,447 ± 0,034
Dresden
0,385 ± 0,038
Fig. 4. Dose response curves of clonogenic survival after X-ray irradiation of normal tissue cell line 184A1 (left) as well as
tumour cell line FaDu (right) with low dose rate (grey curves) and conventional used dose rate (black curves). All curves are
presented in consideration of their 95% confidence intervals.
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L. Laschinsky et al.
Fig. 5. Residual DNA DSB dose response curves obtained after irradiation of normal tissue cell line 184A1 (left) as well as
tumour cell line FaDu (right) with pulsed laser based electron beam (red curves) and continuous electron beam generated by a
therapeutic LINAC (blue curves) after scaling. All curves are presented in consideration of their 95% confidence intervals.
to cell culture conditions at each experimental site compared
to the cell survival and consequently the scaling of the DNA
DSB data was necessary. Hence, the LINAC electron data
were scaled in order to compare with the JETI electron data.
Referring to this the corresponding γH2AX/53BP1 foci
regression data before and after scaling were summarised in
Table 2. In addition, the mean background levels of γH2AX/
53BP1 foci for both cell lines depending on the experiment
location are listed in Table 3.
Figure 5 presents the dose response curves of residual
DNA DSB determined by JETI laser electrons with an ultra
high peak dose rate but low mean dose rate and by clinical
used LINAC electrons after scaling. A reduced biological
effectiveness was obtained for 184A1 cells to laser accelerated electrons compared to clinical used LINAC electrons.
But for FaDu cells again no significant difference was determined for the value of residual DNA DSB caused by the two
electron beams just as seen for cell survival for both cell
lines. The investigation of the mean dose rate effect with
200 kVp X-ray reference irradiation for both dose rate
regimes resulted in no significant difference in the shape of
the dose response curves as well as in the number of residual
γH2AX/53BP1 foci.
The homogeneity and stability of dose delivery by the
laser electron beam was important to guarantee the quality
of the radiobiological results in the present work. For the target areas of the two sample geometries, vessels for cell survival and γH2AX/53BP1 foci, an overall inhomogeneity of
less than 10% was revealed by means of EBT-1 films for all
days and all applied doses. Hence, we could be ensuring that
all cells in each vessel got the same dose and therewith the
radiobiological analyses were well-defined. The accurate
correlation between online and absolute dosimetry as well as
the securing of the correct energy filtering and the control
of the shot-to-shot dose fluctuations have been presented
previously.37) The inter-day dose rate reproducibility and stability of the laser accelerated electron beam over the whole
Fig. 6. Mean dose rates of the electron beam over all cell irradiation of each experimental day at the JETI laser facility. The error
bars indicate the 2σ confidence interval of the values.
cell irradiation campaign is depicted in Fig. 6.
DISCUSSION
The data reported here represent the worldwide first systematic irradiation experiments with normal tissue and
tumour cells using ultra short pulsed laser accelerated electron beams for a comparison with a conventional therapeutic
beam. The JETI laser facility satisfied the basic technical
requirements for radiobiological experiments like beam
reproducibility, stability and reliability of performance over
long times.28,37,43) The available energy of the laser accelerated electrons was already of therapeutic relevance and consequently that electron beam was chosen for the presented
investigation. In order to study the influence of the specific
properties of the novel laser acceleration technology, the
delivery of ultra high peak dose rate due to short pulse duration combined with very high pulse dose, dose response
curves for two cell lines and two biological endpoints were
In Vitro Cell Response to Laser Electrons
investigated. Only in case of 184A1 a reduced biological
effectiveness on DNA DSB for pulsed laser electrons relative to continuous LINAC electrons was determined. In all
other cases, i.e. cell survival for both cell lines and DNA
DSB for FaDu cells, the dose response curves showed no
significant differences in radiobiological effectiveness
between laser accelerated electron beam with an ultra high
peak dose rate and continuous, clinical used LINAC electron
beam.
In order to estimate the influence of the specific very high
peak dose rate of laser accelerated electron beams all other
physical and radiobiological influence factors, like the cell
biological diversity, the energy spectrum with the corresponding LET (linear energy transfer) values and the mean
dose rate of the electron beam had to be excluded or quantified.
The influence of the experimental diversities arising from
different cell culture conditions in the two laboratories were
cancelled out. The non-radiation induced biological effects,
exemplified by the different background level of γH2AX/
53BP1 foci (cf. Table 3), were eliminated by subtraction of
the background levels of the unirradiated control samples.
Moreover, influences which cannot be taken into account by
unirradiated controls have been considered by scaling of the
results using the radiobiological responses of the corresponding 200 kVp X-ray reference irradiation at the two
sites.
To estimate whether the observed difference between
DNA DSB and cell survival in cell line 184A1 is due to
method of γH2AX/53BP1 foci analysis it has to be mentioned that there are several strength and weakness concerning the immunofluorescence staining techniques.42,44,45)
However, all factors should be equal between the experiments performed in Jena and Dresden. Therefore we can
exclude that our method of γH2AX/53BP1 foci analysis
caused the observed reduced biological effectiveness of
184A1 for DNA DSB comparing laser electrons with clinical used electrons.
An influence of the electron spectrum on the results was
minimized by filtering out the biological more effective low
energy JETI electrons. The filtering resulted in a broad, but
downwards limited, JETI electron spectrum of 3–20 MeV,37)
which is characterised by a constant RBE value.46) Furthermore, the clinical used 6 MeV LINAC electrons were in
accordance to the maximum of the energy spectrum of 6.5
MeV of the JETI electrons. Therefore, the influence of the
electron energy spectrum was negligible.
The expected influence of the low mean dose rate with
0.35 Gy/min of the JETI electron beam was ascertained.
This mean dose rate lies within the limits of 0.01 Gy/min to
1 Gy/min, a region where the effects of DNA damage repair,
cell cycle progression and repopulation on the radiobiological effect (i.e. its decreasing) are most pronounced.47–50) This
aspect was studied in detail with 200 kVp X-ray irradiation
401
performed with low dose rate, equal to JETI electrons, compared to conventional dose rate of the LINAC electrons. For
cell survival the modification in the shape of the JETI electron dose response curve to a straight line due to the less
expressed shoulder was confirmed by the 200 kVp X-rays.
This indicated the dose rate effect by low mean dose rate
irradiation which exactly would be expected if there is an
increasing contribution by sublethal damage repair, which
already takes place during the radiation exposure. For residual DNA DSB no significant difference in the shape of the
dose response curves as well as in the mean number of residual DNA DSB was determined for both cell lines (data not
presented). Thus, the observed reduced biological effectiveness of laser electrons compared to clinical used electrons in
the case of 184A1 γH2AX/53BP1 foci is not a result of the
low dose rate effect.
In consequence of the characterisation of the discussed
physical and radiobiological parameters the influence of the
ultra high peak dose rate of laser accelerated electron beam
could be investigated. In the present investigation no significant differences were obtained for comparison of the radiobiological responses due to laser electrons versus clinical
used electrons with the only exception of 184A1 DNA DSB
analysis. Up to now there were no further data for systematic
radiobiological cell responses with precise dosimetry to
laser accelerated electron beams as presented here. In previous studies the issue of different time structures of dose
delivery, especially the ultra high pulse dose rate, which
might result in different radiobiological responses, were
analysed.51–56) Referring to this, studies were performed for
clonogenic survival and γH2AX foci for ultra short pulsed
photons delivered by a laser accelerated plasma X-ray
source with dose rates of up to 1011 Gy/min relative to a conventional photon source, in which no difference was
obtained.51–53) Analysing clonogenic survival following ultra
short pulsed or continuous electron irradiation of conventional radiation sources with single or few nanosecond electron pulses indicates again no significant difference.54,55)
Currently, in vitro studies are performed at the electron beam
of the experimental radiation source ELBE (Electron LINAC
for beams with high Brilliance and low Emittance),57) which
mimic both the ultra high pulse dose rate of the laser electron beam and the conventional pulsed dose rate delivered by
a LINAC. Our first preliminary data indicate no significant
difference in dose responses between ultra high pulse dose
rate and clinical used dose rate.58) These experimental series
as well as the previous studies on conventional radiation
sources confirmed our results that there was no systematic
difference between ultra short pulsed JETI laser electrons
and clinical used LINAC electrons.
The next step in the translational chain includes in vivo
responses to laser accelerated electron beams. Moreover, the
rapid progress in laser technology with increasing the laser
power allows to extent experiments to laser accelerated pro-
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L. Laschinsky et al.
ton beams. By using the new laser system DRACO that provides more than 10 times higher laser power compared to the
actual used JETI laser system allows the acceleration of protons up to 20 MeV for radiobiological experiments.30) First
in vitro cell irradiation experiments have also been performed recently at the J-KAREN laser system with proton
energies up to 2.4 MeV.31)
ACKNOWLEDGMENT
This work was supported by the German Federal Government of Education and Research [03ZIK445].
REFERENCES
1. Combs SE, et al (2010) Particle therapy at the Heidelberg Ion
Therapy Center (HIT) - Integrated research-driven university-hospital-based radiation oncology service in Heidelberg,
Germany. Radiother Oncol 95(1): 41–44.
2. Okada T, et al (2010) Clinical Experiences at National Institute of Radiobiological Science (NIRS). J Radiat Res 51: 355–
364.
3. Bulanov SV, et al (2002) Oncological hadrontherapy with
laser ion accelerators. Phys Lett A 299: 240–247.
4. Ma CMC and Maughan RL (2006) Within the next decade
conventional cyclotrons for proton radiotherapy will became
obsolete and replaced by far less expensive machines using
compact laser systems for the acceleration of the protons. Med
Phys 33: 571–573.
5. Ledingham KWD, Galster W and Sauerbrey R (2007) Laserdriven proton oncology – a unique new cancer therapy? Br J
Radiol 80: 855–858.
6. Malka V, et al (2008) Principles and applications of compact
laser-plasma accelerators. Nature Physics 4: 447–453.
7. Martin M (2009) Laser accelerated radiotherapy: is it on its
way to the clinic? J Natl Cancer Institute 101: 450–451.
8. Kraft G and Kraft SD (2009) Research needed for improving
heavy-ion therapy. New J Phys 11: 025001/1–16.
9. Tajima T and Dawson JM (1979) Laser electron accelerator.
Phys Rev Lett 43: 267–270.
10. Malka V, et al (2002) Electron acceleration by a wake field
forced by an intense ultrashort laser pulse. Science 298: 1596–
1600.
11. Faure J, et al (2004) A laser-plasma accelerator producing
monoenergetic electron beams. Nature 431: 541–544.
12. Geddes CGR, et al (2004) High-quality electron beams from
a laser wakefield accelerator using plasma-channel guiding.
Nature 431: 538–541.
13. Mangles SPD, et al (2004) Monoenergetic beams of relativistic electrons from intense laser-plasma interactions. Nature
431: 535–538.
14. Schwoerer H, et al (2006) Laser-plasma acceleration of quasimonoenergetic protons from microstructured targets. Nature
439: 445–448.
15. Hegelich BM, et al (2006) Laser acceleration of quasimonoenergetic MeV ion beams. Nature 439: 441–444.
16. Pfotenhauer SM, et al (2008) Spectral shaping of laser gener-
ated proton beams. New J Phys 10: 033034/1–14.
17. Fourkal E, et al (2002) Particle in cell simulation of laseraccelerated proton beams for radiation therapy. Med Phys 29:
2788–2798.
18. Fourkal E, et al (2003) Intensity modulated radiation therapy
using laser-accelerated protons: a Monte Carlo dosimetric
study. Phys Med Biol 48: 3977–4000.
19. Malka V, et al (2004) Practicability of protontherapy using
compact laser systems. Med Phys 31: 1587–1592.
20. Luo W, et al (2005) Particle selection and beam collimation
system for laser-accelerated proton beam therapy. Med Phys
32: 794–806.
21. Fourkal E, et al (2007) Energy optimization procedure for
treatment planning with laser-accelerated protons. Med Phys
34: 577–584.
22. Schollmeier M, et al (2008) Controlled transport and focusing
of laser-accelerated protons with miniature magnetic devices.
Phys Rev Lett 101: 0555004.
23. Weichsel J, et al (2008) Spectral features of laser-accelerated
protons for radiotherapy applications. Phys Med Biol 53:
4383–4397.
24. Ma CM, et al (2006) Development of a laser-driven proton
accelerator for cancer therapy. Laser Phys 16: 639–646.
25. Ledingham KWD, McKenna P and Singhal RP (2003) Applications for nuclear phenomena generated by ultra-intense
lasers. Science 300: 1107–1111.
26. Chiu C, et al (2004) Laser electron accelerators for radiation
medicine: a feasibility study. Med Phys 31: 2042–2052.
27. Ledingham KWD and Galster W (2010) Laser-driven particle
and photon beams and some applications. New J Phys 12:
045005/1–66.
28. Beyreuther E, et al (2010) Establishment of technical prerequisites for cell irradiation experiments with laser-accelerated
electrons. Med Phys 37: 1392–1400.
29. Rigaud O, et al (2010) Exploring ultrashort high-energy electron-induced damage in human carcinoma cells. Cell Death
Dis 1: e73.
30. Kraft SD, et al (2010) Dose dependent biological damage of
tumour cells by laser-accelerated proton beams. New J Phys
12: 085003.
31. Yogo A, et al (2011) Measuremant of relative biological
effectiveness of protons in human cancer cells using laserdriven quasimonoenergetic proton beamline. Appl Phys Lett
98: 053701.
32. Linz U and Alonso J (2007) What will it take for laser driven
proton accelerators to be applied to tumor therapy? Phys Rev
ST Accel Beams 10: 094801/1–8.
33. Eicheler W, et al (2002) Splicing mutations in TP53 in human
squamous cell carcinoma lines influence immunohistochemical detection. J Histochem Cytochem 50: 197–204.
34. Menegakis A, et al (2009) Prediction of clonogenic cell survival curves based on the number of residual DNA double
strand breaks measured by γH2AX staining. Int J Radiat Biol
85: 1032–1041.
35. Rangan SRS (1972) A new human cell line (FaDu) from a
hypopharyngeal carcinoma. Cancer 29: 117–121.
36. Stampfer MR and Bartley JC (1985) Induction of transformation and continuous cell lines from normal human mammary
In Vitro Cell Response to Laser Electrons
37.
38.
39.
40.
41.
42
43.
44.
45
46.
47.
epithelial cells after exposure to benzo[a]pyrene. Proc Natl
Acad Sci USA 82: 2394–2398.
Richter C, et al (2011) Dosimetry of laser-accelerated electron
beams used for in vitro cell irradiation experiments. Radiat
Meas 46: 2006–2009.
IAEA (2004) Absorbed dose determination in external beam
radiotherapy: an international code of practice for dosimetry
based on standards of absorbed dose to water. TRS 398. pp.1–
181. International Atomic Energy Agency, Vienna.
Lehnert A, et al (2006) RBE of 25 kV X-rays for the survival
and induction of micronuclei in the human mammary epithelial cell line MCF-12A. Radiat Environ Biophys 45: 253–260.
Beyreuther E, et al (2009) DNA double-strand break signalling: X-ray energy dependence residual co-localised foci of
γH2AX and 53BP1. Int J Radiat Biol 85: 1042–1050.
Richter C, et al (2009) Energy dependence of EBT-1 radiochromic film response for photon (10 kVp-15 MVp) and electron beams (6-18 MeV) readout by a flatbed scanner. Med
Phys 36: 5506–5514.
Leatherbarrow EL, et al (2006) Induction and quantification
of gamma-H2AX foci following low and high LET-irradiation. Int J Radiat Biol 82: 111–118.
Pawelke J, et al (2009) Laser particle acceleration for radiotherapy: a first radiobiological characterization of laser accelerated electrons. IFMBE Proceedings, Berlin, Heidelberg,
Germany: Springer 25(III): 502–504.
Scherthan H, et al (2008) Accumulation of DSBs in γH2AX
domains fuel chromosomal aberrations. Biochem Biophys Res
Communications 371: 695–697.
Löbrich M, et al (2010) γH2AX for analysis for monitoring
DNA double-strand breaks repair – strength, limitations and
optimization. Cell Cycle 9: 662–669.
Hunter N and Muirhead CR (2009) Review of relative biological effectiveness dependence on linear energy tranfer for lowLET radiations. J Radiol Prot 29: 5–21.
Hall EJ (1972) Radiation dose-rate: a factor of importance in
radiobiology and radiotherapy. Br J Radiol 45: 81–97.
403
48. Mitchell JB, Bedford JS and Bailey SM (1979) Dose-rate
effects in plateau-phase cultures of S3 HeLa and V79 cells.
Radiat Res 79: 552–567.
49. Steel GG, et al (1986) Dose-rate effects and the repair of radiation damage. Radiother Oncol 5: 321–331.
50. Kreder NC, et al (2004) Cellular response to pulsed low-dose
rate irradiation in X-ray sensitive hamster mutant cell lines. J
Radiat Res 45: 385–391.
51. Tillmann C, et al (1999) Survival of mammalian cells exposed
to ultrahigh dose rates from a laser-produced plasma X-ray
source. Radiology 213: 860–865.
52. Shinohara K, et al (2004) Effects of single-pulses (≤ 1 ps) xrays from laser-produced plasmas on mammalian cells. J
Radiat Res 45: 509–514.
53. Sato K, et al (2010) γ-H2AX and phosphorylated ATM focus
formation in cancer cells after laser plasma X irradiation.
Radiat Res 174: 436–445.
54. Nias AHW, et al (1970) Survival of HeLa cells from 10 nanosecond pulses of electrons. Int J Radiat Biol 17: 595–598.
55. Gerweck LE, et al (1979) Repair of sublethal damage in mammalian cells irradiated at ultrahigh dose rates. Radiat Res 77:
156–169.
56. Schmid TE, et al (2009) No Evidence for a different RBE
between pulsed and continuous 20 MeV protons. Radiat Res
172: 567–574.
57. Zeil K, et al (2009) Cell irradiation setup and dosimetry for
radiobiological studies at ELBE. Nucl Instrum Meth Phys Res
B 267: 2403–2410.
58. Laschinsky L, et al (2009) Systematic in vitro cell experiments with laser accelerated electron beams. Radiother Oncol
92(Suppl.1): S89.
Received on May 6, 2011
Revision received on November 10, 2011
Accepted on January 19, 2012
J-STAGE Advance Publication Date: May 11, 2012