www.nature.

com/scientificreports

OPEN Fast dose fractionation using

ultra-short laser accelerated
proton pulses can increase cancer
Received: 25 February 2019
Accepted: 28 June 2019 cell mortality, which relies on
functional PARP1 protein
Published: xx xx xxxx

E. Bayart 1, A. Flacco1, O. Delmas1, L. Pommarel1,2, D. Levy1,3, M. Cavallone1,

F. Megnin-Chanet4,5, E. Deutsch 4 & V. Malka1,3
Radiotherapy is a cornerstone of cancer management. The improvement of spatial dose distribution in
the tumor volume by minimizing the dose deposited in the healthy tissues have been a major concern
during the last decades. Temporal aspects of dose deposition are yet to be investigated. Laser-plasma-
based particle accelerators are able to emit pulsed-proton beams at extremely high peak dose rates
(~109 Gy/s) during several nanoseconds. The impact of such dose rates on resistant glioblastoma
cell lines, SF763 and U87-MG, was compared to conventionally accelerated protons and X-rays. No
difference was observed in DNA double-strand breaks generation and cells killing. The variation of the
repetition rate of the proton bunches produced an oscillation of the radio-induced cell susceptibility
in human colon carcinoma HCT116 cells, which appeared to be related to the presence of the PARP1
protein and an efficient parylation process. Interestingly, when laser-driven proton bunches were
applied at 0.5 Hz, survival of the radioresistant HCT116 p53−/− cells equaled that of its radiosensitive
counterpart, HCT116 WT, which was also similar to cells treated with the PARP1 inhibitor Olaparib.
Altogether, these results suggest that the application modality of ultrashort bunches of particles could
provide a great therapeutic potential in radiotherapy.

Among radiotherapy modalities, the use of hadrons (i.e. protons and heavier ions) for malignant tumor treatment
takes advantage from their energy transfer characteristics in comparison to widely used X-rays. Hadron therapy
allows high conformation of the dose distribution to the target volume because of the sharp lateral penumbra and
distal dose fall-off, as most of the energy is deposited within the Bragg peak. These properties make it possible to
increase dose deposited in the tumor target while reducing dose in adjacent healthy tissues, preventing associated
sides effects1,2.
During the past decade, important goals have been reached on laser-driven proton sources, making them a
reliable alternative for in-vitro studies. The biggest difference between laser-driven sources and conventional ones
is the temporal structure of the irradiation. While conventional proton sources deliver a continuous beam at a
dose-rate of several Gy/min, laser-driven particle beams are delivered as separate ultra-short bunches, typically
in the range of nanoseconds, and dose rates as high as 109 Gy/s3–5. Laser sources at Hz repetition rates are hence
capable of delivering comparable average dose rates, whereas peak dose rate is 6 to 9 orders of magnitude higher.
While development of laser-driven proton sources is still ongoing, to reach energies relevant for clinical
applications, it is crucial to characterize the radiobiological effects of pulsed ionizing radiation at high dose rate.

1
LOA (ENSTA/CNRS/École Polytechnique), Sources for Irradiation, Imaging and Medical Applications, Institut
Polytechnique de Paris, 828 Bd des Maréchaux, 91762, Palaiseau, CEDEX, France. 2Present address: Airbus GmbH,
Claude-Dornier-Straße, 88090, Immenstaad, Germany. 3Department of Physics of Complex Systems, Weizmann
Institute of Science, Rehovot, 7610001, Israel. 4INSERM 1030, Univ Paris-Sud, Univ Paris-Saclay, Department of
Radiation Oncology, Gustave Roussy Cancer Campus, Villejuif, France. 5Present address: INSERM U1196/UMR9187,
CMIB - Institut Curie, bât 112, Centre Universitaire, 91405, Orsay, France. Correspondence and requests for materials
should be addressed to E.B. (email: [email protected])

Scientific Reports | (2019) 9:10132 | https://doi.org/10.1038/s41598-019-46512-1 1

www.nature.com/scientificreports/ www.nature.com/scientificreports

Figure 1. Dose responses of DNA damage foci formation and of cell survival. (A) Representative immune-
fluorescent images of SF763 cells obtained 1 h and 24 h after exposure to the indicated doses of laser driven
protons. The negative controls (0 Gy) were sham-irradiated. Merged images show γH2AX (green) and DNA
(blue). (B, C) Number of γH2AX foci induced by laser driven protons (LDP, dotted square), conventional
accelerated protons (CAP, triangles) and X-rays (x cross) respectively in SF763 and U87-MG cells. Each data
point represents the mean of at least three independent experiments in which at least 300 nuclei were analyzed
and averaged. (D, E) Normalized cell survival resulting from exposure to increasing doses of LDP, CAP and
X-rays respectively in SF763 and U87-MG cells. Each data point represents the mean and standard deviation
(SD) of three replicates obtained at least with three independent experiments. Survival curves were generated
following the linear quadratic model (R > 0.96 and R > 0.97 for SF763 and U87-MG cell lines respectively).

Although the biological effects of proton irradiation on living systems have been widely studied6, much still has
to be explored on the impact of protons delivered in such short pulses of ultra-high dose rates on living cells or
tissues. During the last decade, several experimental campaigns proved the feasibility of radiobiological studies
on intense laser facilities and were able to evaluate the biological effectiveness of such beam3–5,7–13. These studies
suggest that the radiobiological effectiveness of laser-driven protons is roughly similar to conventional beams,
when considering DNA damaging potential or tumor cell killing. We recently described a set-up of four perma-
nent magnet quadrupoles to shape and control the proton beam generated by the multi-terawatt laser SAPHIR at
LOA, the only (French) laser-plasma infrastructure dedicated to radiobiology studies, and validated the robust-
ness of the system by irradiating radiosensitive colorectal cancer cell line5. Here we confirmed the efficiency of
laser-plasma proton beams compared to conventional ones on radioresistant glioblastoma cell lines, for which
proton therapy is indicated. As a further step, we investigated the biological impact of the temporal aspect of
laser-driven proton bunches. Despite the challenging implementation of radiobiology assays on a laser facility,
we show that the variation of proton bunch repetition rate is associated with an oscillation of cell survival, which
is found to be dependent on the PARP1 (poly ADP-Ribose polymerase 1) protein activity in tumor cells. This is
the very first time the temporal structure of laser beam, that we called fast dose fractionation, is investigated and
demonstrated to provide an increased therapeutic potential.

Results
Laser driven protons (LDP) are as efficient as conventional accelerated protons (CAP) and
X-rays in inducing DNA double strand breaks and cell killing on glioblastoma cell lines. The
favorable ballistic of proton beams makes such treatment efficient for brain, base-of-skull and head-and-neck
tumors. As previous experiments were performed on rodent, HeLa, lung or colorectal cells3–5,7,8,10,11, we decided to
study the impact of LDP on the highly resistant glioblastoma cells lines, SF763 and U87-MG, with regard to CAP
or X-rays. We first compared LDP-induced DNA double strand breaks (DSBs). DSBs were detected by micros-
copy through immunodetection of the histone H2AX phosphorylation on Ser139 (γH2AX). Cells were fixed
one or 24 hours after three and six LDP’s bunches (corresponding to 2.1 ± 0.42 and 4.2 ± 0.84 Gy respectively,
see methods section) delivered every 30 seconds, or two and four Gy of CAP or X-rays (Fig. 1A). As expected
the amount of DSBs increased as the dose increased; it decreases between 1 h and 24 h, which reflects the DNA
repair process. Similar amount of foci were quantified for LDP, CAP or X-rays one hour after irradiation either in

Scientific Reports | (2019) 9:10132 | https://doi.org/10.1038/s41598-019-46512-1 2

www.nature.com/scientificreports/ www.nature.com/scientificreports

Radiation SF763 U87-MG

LDP 7.45 ± 0.31 7.47 ± 0.32
CAP 7.90 ± 0.18 7.11 ± 0.16
X-rays 7.95 ± 0.24 7.45 ± 0.06

Table 1. Comparison of doses giving 10% of cell survival (D10 values in Gy) from LDP, CAP and X-rays. D10
mean values ± SEM extracted from curves obtained in Fig. 1D, E are reported. Each value represents the mean
of at least three independent experiments. Comparison using by two way ANOVA multiple comparisons test
(Tukey’s multiple comparisons test) gave, at least, p > 0.33.

SF763 or U87-MG cells (Fig. 1B, C). Twenty four hours post-irradiation, no differences were observed for SF763
while the number of residual foci seemed to be higher for LDP compared to CAP and X-rays in U87-MG cells.
However, this apparent difference was not significant. These results suggest that LDP induce DNA double strand
breaks in resistant glioblastoma cell lines as well as CAP or X-rays.
To follow-up on the radiobiological consequences of LDP on cancer cells, we evaluated next how cell survival
was affected by LDP. For that purpose, the two glioblastoma cell lines were irradiated with 0 to 12 proton bunches
(corresponding to doses ranging from 0 up to 10 Gy) of LDP and the resulting dose response survival curves
obtained from non clonogenic survival assays (see methods section) were compared to those obtained with cells
irradiated with CAP or X-rays. It can easily be noticed that, for the SF763 cells, the survival curves corresponding
to each irradiation condition overlap (Fig. 1D), giving similar D10 (doses giving 10% of cell survival, p values were
>0.3, Table 1); the same result was observed with the U87-MG cell line. These data indicate that the impact of
LDP on cell survival is similar to the one of CAP or X-rays.
These results show that no significant difference in DNA double strand breaks detection and cell survival
measurement could be detected between LDP, CAP and X-rays in highly resistant glioblastoma cell lines. These
observations are in agreement with previous studies using others cell lines3–5,7,8,10,11 and confirm that laser-driven
protons are able to affect cancer cells, at the global DNA damage and cell survival levels, with the same effective-
ness compared to conventional accelerated particles.

Changing the dose delivery modality by modifying the repetition rate of LDP’s bunches can
result in cell survival oscillation. Laser-driven proton acceleration in our configuration (see methods)
reaches a maximum repetition rate of 0.5 Hz, i.e. a minimum of 2 seconds between two consecutive proton
bunches. This delay can be increased to any value with a resolution of 200 ms. In our experiment, the cell lines
under test (glioblastoma SF763 and U87-MG, colorectal cancer cells HCT116-WT and HCT116-p53−/−) were
exposed to a fixed number of LDP bunches (five or nine for colorectal cancer and glioblastoma cell lines respec-
tively). Different samples were irradiated with a variable delay between shots, from 60 to 2 seconds. Hence the
total irradiation time varied from 15 to 540 seconds. Cell survival of each cell lines was monitored and reported
as a function of the delay between LDP’s bunches.
As shown in Fig. 2, such variation did not affect the survival neither of SF763 cells (p > 0.531, Fig. 2A) nor of
U87-MG cells (p > 0.222, Fig. 2B). In the case of HCT116 cells, variation of proton bunches cadency affected the
cell survival. The same outcome was observed with both HCT116 WT and its radioresistant counterpart HCT116
p53−/−; as expected the survival of HCT116 p53−/− cells remained higher than the WT. For the two cell lines
however the observed surviving fraction was a non-monotonic function of the delay between each pulse, with
the apparition of two different maxima (p ≤ 0.0038 and p < 0.0001 for HCT116 WT and HCT116 p53−/− respec-
tively). Interestingly, the cell survival difference between HCT116 p53−/− and HCT116 WT was maximum when
the curves reached their maxima, then the two converged together at the shortest delays (at 2 s p = 0.9982). This
observation suggests that an increase of proton bunch cadency could lead, depending on cell type, to an increase
in cell mortality.

Non-monotonic character of cell survival as a function of LDP proton bunches cadency is

related to the presence of functional PARP1 protein. About two decades ago, it was observed that
cells exposed to two brief pulses of relativistic electrons exhibited variable survival rates as a function of the delay
between the two pulses. This phenomenon is called W-effect (WE) from the shape of the survival rate oscillation,
where the delay was varied between few minutes and sub-second14. This process appeared to vary considerably
among different cell lines and was shown to be dependent on an active PARP1 (poly(ADP-ribose) polymerase)
protein15. The variation of the cell survival we observed in HCT116 cells by varying LDP proton bunch repetition
rate could share origins with the W effect. However, this non-monotonic behavior was not observed in the other
tested cell lines, which seems to indicate that this variation of cell susceptibility could also be linked to the PARP1
protein. Oxidative stress promotes the formation of large amounts of base damage leading to DNA breaks, which
induces the over-activation of PARP1 and leads to protein parylation16. Thus, HCT116, SF763 and U87-MG cells
were exposed to hydrogen peroxide (H2O2), then Western-Blot analyses were performed to detect the PARP1
protein and the related parylation. As shown in Fig. 3A, the PARP1 protein was detected in HCT116 WT cells as
well as in HCT116 p53−/− cells. When exposed to H202, parylation was induced, demonstrating the functional
activity of PARP1 in these two cell lines. On the contrary, in U87-MG cells, very low amount of PARP1 could be
detected and only extremely weak parylation was observed. Surprisingly, in SF763 cells, the PARP1 protein was
efficiently detected, however, no increase of parylation was observed after cell exposition to hydrogen peroxide.
This result suggests that in this cell line, although present, the PARP1 protein was not functional hence not able to
promote parylation. The PARP1 protein activation and the related parylation process are crucial for DNA damage

Scientific Reports | (2019) 9:10132 | https://doi.org/10.1038/s41598-019-46512-1 3

www.nature.com/scientificreports/ www.nature.com/scientificreports

Figure 2. Cell survival dependency to the variation of the bunch repetition rate. Normalized survival fraction
resulting from exposure for a given dose of LDP bunches delivered with different delay ranging from three to
60 seconds. Radioresistant Glioblastoma cell lines SF763 (triangles) (A) and U87-MG (dots) (B) were submitted
to a dose corresponding to nine bunches of LDP (6.3 ± 1.39 Gy). (C) radiosensitive colorectal cancer HCT116
cells, WT (white squares) and p53−/− (black squares) were exposed to five bunches of LDP (3.5 ± 0.77 Gy). Each
data point represents the mean and SD of three replicates obtained in at least three independent experiments.

repair pathways activation17. Inhibition of PARP1 in cells where PARP1 is active produces an increased sensitivity
to genotoxic stress, particularly in response to ionizing radiations18. To exploit this advantage for the treatment of
cancer, PARP1 inhibitors, such as Olaparib have been developed19.
To further correlate the PARP1 activity status to the oscillating cell susceptibility to variation of the cadency
of shots, cells from each of the cell lines previously used were treated or not with Olaparib (200 nM) and then
subjected to increasing dose of X-rays. The corresponding survival curves were generated (Fig. 3B, C) and D10
values (doses giving 10% of cell survival) were extracted and compared (Fig. 3D). As expected, in HCT116 WT
cells, where PARP1 is functional, the use of Olaparib significantly increased cell mortality, which resulted in
a decreased D10 values: from 5.29 ± 0.37 to 4.38 ± 0.28 (p = 0.0039). The same outcome was observed treating
HCT116 p53−/− cells for which D10 value went from 5.95 ± 0.36 down to 5.29 ± 0.30 (p = 0.0082). On the con-
trary, no significant variation of cell survival was detected with SF763 and U87-MG cells, exhibiting no or very
few PARP1 activity, when submitted to the same treatment (D10 = 8.70 ± 0.29 without and D10 = 8.45 ± 0.52 with
Olaparib treatment in SF763 cells, p = 0.3558; D10 = 8.07 ± 0.55 without and D10 = 7.72 ± 0.48 with Olaparib
treatment in U87-MG cells, p = 0.1166). Together, these results suggest that the cell survival variation with the
proton bunch cadency is related to the presence of a functional PARP1 protein.

An increase of LDP’s bunches cadency can promote same cell radiosensitization as the use of
PARP1 inhibitor. Cell survival level in response to variation of proton bunches repetition rate was correlated,
in the different cell lines, to the presence of a functional PARP1 protein. It was verified next whether an inhibi-
tion of PARP1 would affected this variation or not. For this purpose, HCT116 WT cells were exposed, as in the
previous experiment, to Olaparib (200 nM) to inhibit PARP1, which led, as shown by western blot (Fig. 4A), to
an absence of the H2O2-induced parylation. Cells were then subjected to five LDP’s bunches with delay ranging
from 3 to 45 seconds.
As expected, in absence of Olaparib, HCT116 WT cell survival variation was non-monotonic, increasing
between 25 and 45 seconds to reach a maximum (p ≤ 0.0316) before decreasing from 25 to 2 seconds where it
reached a minimum. On the contrary, when cells were incubated in presence of Olaparib before exposition to
LDP, cell survival variation was not detected any more (p ≥ 0.0754). Interestingly, when bunches were separated
by 2 seconds, the cell survival of cells only irradiated by LDP’s bunches reached the level obtained by those sub-
mitted to Olaparib treatment before LDP exposure, giving equivalent values (p > 0,9999). This result first demon-
strates that non-monotonic cell survival depends on the PARP1 protein activity. The PARP1 protein is a crucial
actor in DNA damage signaling20, suggesting that cell survival variation induced by the LDP bunch repetition
rate could be due to a specific impact of this dose deposition modality on DNA damage signaling and repair.
Furthermore, we observed that an increase of the proton bunch cadency led cells exhibiting a functional PARP1
activity to the same decrease of cell survival level as combining LPD to PARP1 inhibition: this suggests that only
using specific LDP dose deposition modality could be as efficient as drug-combined radiotherapy.

Discussion
The main finding of this study is that more than the dose itself, the temporal dose deposition modality of LDP is
a key feature, determining cancer cells response. Because of their favorable ballistic properties, hadrons (protons
and carbon ions) represent a better alternative for the radiation therapy of solid cancer affecting organ-at-risk1.
Due to the Bragg pick dose deposition profile, the dose is focused inside the target volume, reducing side effects
to the neighboring healthy tissue. The development of laser plasma technology and its new paradigms for protons
acceleration21 raise new possibilities for studying the effects of dose delivery modalities22–24.
Since the first proof of concept of irradiating cells with LDP8,12, less than ten years ago, we and others have
studied the biological impact of laser-accelerated protons and shown similar effectiveness of LDP, compared to
conventional beams, to generate DSBs3,10 and to induce cell killing4,5,9,11. None of the previously existing studies,
performed on various animal and human cell types, included highly resistant glioblastoma cells, one of the main

Scientific Reports | (2019) 9:10132 | https://doi.org/10.1038/s41598-019-46512-1 4

www.nature.com/scientificreports/ www.nature.com/scientificreports

Figure 3. Determination of PARP1 status and related radiosensitization. (A) Western Blot detection of the
PARP1 protein and parylation in total protein extracts from HCT116 WT, HCT116 p53−/−, SF763 and U87-MG
cells left untreated or treated 10 min with 1 mM hydrogen peroxide (H2O2). Protein amounts were normalized
probing membrane with an anti-GAPDH antibody. (B) survival curves obtained after exposure of SF763
(triangles) and U87-MG (diamonds) cells left untreated (opened symbols) or treated with 200 nM Olaparib
(filled symbols) to increasing doses of X-rays. Survival curves were generated following the linear quadratic
model (R > 0.96). Each points represents the mean and SD of at least three independent experiments. (C) As for
(B) with HCT116 WT (squares) and HCT116 p53−/− cells (circles). (D) D10 values determined from survival
curves depicted in (B, C) where white bars correspond to cells left untreated and filled bars to cells treated with
200 nM Olaparib. D10 mean values ± standard error of mean (SEM) are represented and were compared for each
cell line using ratio paired t test.

indications of proton therapy. We observed, in agreement with the literature, similar DNA damaging potential
and cell survival in response to LDP or conventional accelerators.
We and others have however reported a number of non-significant divergences which were considered to arise
from differences in experimental procedures and endpoints (e.g. the dose applied), in reference photons and pro-
tons energies and LDP spectra. This study suggests that such discrepancies could also depend on the applied dose
deposition modalities. When a sample is irradiated by laser-generated particles (electrons or protons), the total
deposited dose is delivered as a sequence of multiple ultra-short bunches at ultra-high dose rates; the dose per
bunch, hence the number of bunches required for a given dose, and the effective repetition rate depend on tech-
nical choices at the facility3–5,7–13. Our study explores the radiobiological impact of the repetition rate of bunches
and suggests that parameters such as the dose-per-bunch and the distribution in time of the LDP bunches should
be better taken in account in future studies.
By varying the delay between proton bunches, we observed a non-monotonic variation of cell survival in
HCT116 cells; this variation was not observed in SF763 nor in U87-MG cell lines. Similar variations with low
energy electrons were already reported, two decades ago. Indeed, it has been shown, using a linear accelerator,
that pulse irradiation brings about multiphasic, synchronous changes in the susceptibility of cells to a second
pulse of radiation applied at variable intervals, lasting from fractions of second to a few minutes: these changes
are at the origin of the W-shaped survival curve (W-effect)14,25. Further exploration of this phenomenon revealed

Scientific Reports | (2019) 9:10132 | https://doi.org/10.1038/s41598-019-46512-1 5

www.nature.com/scientificreports/ www.nature.com/scientificreports

Figure 4. Impact of PARP1 inhibition on cell survival oscillation in response to the variation of the delay
between LDP bunches. (A) Western Blot detection of the PARP1 protein and parylation in total protein extracts
from HCT116 WT cells left untreated, treated 10 min with 1 mM hydrogen peroxide (H2O2) or treated one
hour with 200 nM Olaparib before H2O2 treatment. (B) HCT116 WT cells were left untreated (white circles)
or treated one hour with 200 nM Olaparib (black circles) and were then exposed to five bunches of LDP
(3.5 ± 0.77 Gy). Each data point represents the mean and SD of three replicates obtained in two independent
experiments. Comparisons of the cell survival were performed using by two way ANOVA multiple comparisons
test (Tukey’s multiple comparisons test).

that the WE depends on a functional PARP1 protein15. In our experiment, we also observed that the response of
cell survival induced by the variation of the LDP bunches cadency was correlated to the presence of PARP1 and
an efficient parylation process in cells exposed to H2O2. Hydrogen peroxide results in strong oxidative stress in
cells, promotes the formation of large amount of base damage, leading to DNA breaks and protein parylation,
which mostly accounts for PARP116. Protein parylation in response to genotoxic stress is essential to promote
DNA damage signalization and activation of DNA repair pathways17,26. Consequently, the inactivation of the
PARP1 results in enhanced genomic instability and increased radiation sensitivity18. This mechanism has been
already exploited to develop drugs for cancer treatment. Several PARP inhibitors, such as Olaparib (AZD-2281,
ASTRA-ZENECA), are currently tested in the clinic in combination with radiotherapy27. Consistently, in cells
exhibiting normal parylation and PARP1 protein amount (HCT116 cells), the use of Olaparib combined with ion-
izing radiations led to an increased cell susceptibility that was not observed in SF763 and U87-MG, in which par-
ylation was impaired. This existing parylation default explains why the U87-MG cell line was previously reported
to be a non-responder cell line to PARP inhibitor28,29. These results are consistent with the very recently published
results showing that the deletion of PARP1 protein leads to resistance to PARP inhibitors30. They also explain
why no difference of cell survival was observed for those cells when irradiations were performed with continuous
beams. We then showed that, as for the WE, the bunches cadency related cell survival variation was abolished
in response to PARP1 inhibition using Olaparib. This observation supports the idea that those two phenomena
are related to DNA damage signaling or repair pathways. Indeed, it has been suggested that irradiation of cells at
critical times of DNA damage recognition or repair following the first irradiation exposure may promote chro-
mosome damage and long-lasting genomic instability14,15. We did not observe any significant difference between
either the early or the late amounts of DNA double strand breaks (DSBs) induced by LDPs to conventional beams
in Glioblastoma cell lines. However, although it has been shown that PARP-1 is able to interact with proteins
involved in DSBs repair pathways, non-homologous end joining31 or homologous recombination32, the PARP-1
protein is essential for base excision repair (BER) pathway. Deposition of large amounts of particles in a very short
time and with a high repetition rate could result in an increased formation and accumulation of complex DNA
damage and/or in DNA damage clusters that could not have been detected performing standard DSBs analysis
with a repetition rate leading to a mean dose rate similar to conventional beams (Fig. 1B, C). This particular
mechanistic point will be deeper explored in future experimental campaigns.
The W-effect was demonstrated by irradiating cells with electron bunches at a dose rate of 12 Gy/s14,15. Our
observation, consistent with W-Effect and obtained with proton bunches, suggests this phenomenon to be likely
related to the temporal feature of the dose delivery rather than the particles. The W-effect was observed after
the irradiation with only two electron bunches. In our experimental conditions, excepted for the lowest dose of
dose-response survival curves, the application of several pulses was required to reach the desire total dose. In
previously published results with laser accelerators, dose was deposited either in one shot3,4 or applying tens of
shots depending on the available dose per shot of the infrastructure used9–13. As variation of cell survival appears
to be correlated to multiple bunch irradiation, no differences could be observed with regards to conventional irra-
diation when cells were irradiated using a single bunch. In the case of dozen of bunches, the dose per bunch was
very low ranging from 0.03 to 0.3 Gy per shot9–13. W-effect has been described applying shots of 1, 2 or 4 Gy14,15
and, in our study, the SAPHIR parameters were set up to deliver a dose per bunch in the range of 1 Gy. These

Scientific Reports | (2019) 9:10132 | https://doi.org/10.1038/s41598-019-46512-1 6

www.nature.com/scientificreports/ www.nature.com/scientificreports

observations suggested that the variation of cell susceptibility in response to multiple bunch irradiation occurs
only if each single shot bunch has a minimal dose close to the Gray or more, leading sufficient DNA damage and
PARP1 activation.
In our experimental conditions, the cell survival reached its maximum when the delay between bunches was
about 25 s. This result explains why no differences in cell survival was found between LDP and conventional
beams in our previous study, with a repetition rate of about 1 shot every 30 seconds5, leading to an average
dose rate in the range of 1.4 Gy/min, roughly as much as conventional beams. This value probably relies on the
intrinsic susceptibility of HCT116 cells and may be different with different cell types. Indeed, it has been shown
that the W-shaped survival curve profile varies from a cell line to another, with the “peak” occurring between
3 and 60 seconds14,15. We did not observe a W-shaped curve but a bell-shaped curve when varying the pace of
bunches. We cannot exclude that with higher repetition rate, cell survival could increase again. This condition
will be explored in a next future, when the LDP generation will be upgraded to reach a repetition rate higher than
0.5 Hz. It could also come from the fact that we applied sequences of five LDP bunches on HCT116 cells whereas
WE was observed with two bunches. Either the application of more than two short pulses of ionizing radiations
could result in the modification of the W-curve profile or it could induce some kind of resonance of the biological
mechanisms involved, resulting in a kind of bell-shaped profile. These results suggest that fast fractionation of the
dose has an additional impact on the resulting cell survival at constant dose, whose effect varies depending on the
numbers and the repetition rate of pulses. The W-effect is the consequence of a particular fast dose fractionation
condition, the application of two pulses, whereas the application of several pulses leads to non-monotonic cell
survival variation.
Finally, we observed the lowest cell survival for a bunch delay of about 2-3 seconds. Interestingly, at this rep-
etition rate, the cell survival of the radioresistant HCT116 p53−/− cells converged to that of HCT116 WT cells,
which was similar to the case when Olaparib was added. This result suggests that, depending on the bunch repe-
tition rate, cell killing obtained with laser pulsed particles could be as efficient as combined therapy using PARP
inhibitors. This particular point highlights a therapeutic advantage that laser plasma accelerators could provide
compared to continuous therapeutic beams. Indeed, the use of PARP inhibitors in clinical protocols leads, as any
drug, to side effects and drug resistance33,34. Altogether, these results demonstrate for the first time the proof of
concept of a radiobiological advantage to perform dose deposition applying successive bunches of ultra-high dose
rate particles such as protons. Further investigations are required to precise the conditions allowing the fast frac-
tionation effect to be observed as e.g. the minimal or maximal total dose to be delivered, and to support the great
therapeutic potential of laser plasma technology and pulsed beams of ionizing radiation to provide innovations
in radiotherapy for cancer treatment.

Methods
Cell culture and chemical treatments. The human colorectal cancer HCT116 WT and p53−/− cells were
cultured in McCoy’s5A (Modified) Medium. The human glioblastoma cell lines, SF763 and U87-MG were cul-
tured in Dulbecco’s modified Eagle’s minimum medium with Glutamax (Thermo Fisher Scientific). Cells were
grown as monolayers in their respective medium, supplemented with 10% fetal calf serum (PAA) and 1% pen-
icillin and streptomycin (ThermoFisher Scientific) in plastic tissue culture disposable flasks (TPP) at 37 °C in
a humidified atmosphere of 5% CO2 in air. For PARP1 inhibition, cells were incubated one hour in medium
supplemented with Olaparib (AstraZeneca, 20 µM diluted in DMSO) to a final concentration of 200 nM before
irradiation or oxidative treatment. Untreated cells were exposed to the same volume of DMSO. For hydrogen per-
oxide (H2O2) treatment, H2O2 (Sigma Aldrich 216763, 100 mM in water) was added to culture medium to a final
concentration of 1 mM and cells were left exposed for a 10 min incubation time at room temperature.

Conventional irradiation conditions. X-ray irradiations were performed using a Varian NDI 226 × -ray
tube applying as previously described35 a tension of 200 kV and an intensity of 15 mA with a dose rate of 1,23 Gy/
min.
Irradiation with conventional accelerated protons (CAP) were performed using the proton beam at the Curie
Institute Proton Therapy Center36: an IBA C230 isochronous cyclotron, which generates a proton beam with
an initial energy of 235 MeV. The beam energy is lowered to 201 MeV right at the cyclotron output and further
reduced down to 20 MeV by sequential polycarbonate and Plexiglas attenuators.

Laser-driven proton source. The LDP irradiations were performed with laser-driven, TNSA-based proton
beam. In a TNSA (Target Normal Sheath Acceleration) proton source, protons are accelerated by the charge sep-
aration that is produced at the frontier of an expanding plasma. Such a plasma is created and heated during the
interaction between an intense, ultra-short laser pulse and a solid target, where the laser field at high intensity effi-
ciently heats electrons in the target material and increases the accelerating field, hence the proton final energy21.
In our experiments, the 200 TW laser source of SAPHIR (Amplitude Technologies) at the Laboratoire d’Op-
tique Appliquée (LOA, Palaiseau, France) was used as a main driver. The laser pulse duration is 26 fs and the laser
energy on target is 3 J per shot, for an on-target intensity approaching 1020 W/cm2. The laser pulse was focused
on 5 μm thick Titanium targets, which provided the bulk material for the production of a plasma and the surface
where hydrogen ions sit before acceleration. A target is destroyed at each shot. A semi-automatic alignment pro-
cedure ensures the alignment of a fresh target after each shot within tolerances, in order to ensure comparable
acceleration conditions during the multi-shot irradiation procedure. The initial laser repetition rate was thus
lowered from native 5 Hz to single shot, in order to match the pace of the target change, which can be slower or
equal to 0.5 Hz (2 seconds between two consecutive shots).
The particle beam shaping, selection and transport was ensured by a set of four permanent magnet quadru-
poles37. TNSA-based sources produce a multi-specie, highly divergent, laminar and chromatic particle beam. The

Scientific Reports | (2019) 9:10132 | https://doi.org/10.1038/s41598-019-46512-1 7

www.nature.com/scientificreports/ www.nature.com/scientificreports

Figure 5. LDP quality control. Proton spectra recorded by RCF films (integration over the entire emission
cone) before (A) and after (B) the magnetic transport. (from Pommarel et al.5). (C) Shot-to-Shot fluctuation
analysis. Each dot represent a single shot (n = 3504). Mean and SD are represented (red lines).

transport line has been designed to reject electrons, unwanted ion species and to focus the proton emission to a
surface of 2 cm2 where the biological samples were positioned, 97 cm far from the plasma point. Proton energy
spectrum before and after the magnetic transport are provided in Fig. 5A, B respectively.
The quality of the beam during target irradiation (shot-to-shot stability and total deposited dose) was assessed
by real-time dosimetry measurements. These are obtained through a transmission ionization chamber (PTW 786,
100 μm water equivalent thickness); the ionization chamber combined with an electrometer (PTW, UNIDOS E)
was previously calibrated with the synchrotron proton beam at the Orsay Protontherapy center (CPO).
®
Dosimetry measurements in the conditions used in the present study showed a total mean dose-per-shot of
0.72 ± 0.17 Gy (Fig. 5C) within the 2 cm2 irradiation surface. The dose map per unit surface has a uniformity of
20% rms. Laser-accelerated particle bunches hold the temporal signature of the fast accelerating process, which
takes place during a time shorter than 1 ps. In the case of MeV protons, the continuous spectrum produces a
stretch of the particle bunch during its propagation. Considering the distance between the proton emission point
and irradiation point, as well as the available spectrum, total dose in a single shot was delivered at the biological
target during τ = 4.8 ns. This corresponded, for a measured dose of 0.7 Gy/shot, to a peak dose rate in the order
of 1.5 × 108 Gy/s5.

®
Cell survival assay. The cell containers used for irradiation were lumox dish 35 (SARSTEDT) exhibiting a
®
25 µm thick lumox bottom face. Depending on beam transverse profile and position observed on a sensitive film
®
(Image plate, Fujifilm) a 18 mm × 0.9 mm area was delimited on the internal face of the lumox membrane, where
6 × 104 cells were seeded. Cells were let grow overnight in 200 µl of medium. To generate dose-response survival
curves, HCT116 cells were exposed to either 1, 2, 4, 6 or 8 consecutive bunches of LDP or to 1, 2, 3, 4 or 6 Gy of
CAP or X-rays, whereas SF763 and U87-MG cell lines were subjected to 2, 5, 7, 10 or 12 consecutive bunches of
LDP or 2, 4, 6, 8 or 10 Gy of CAP or X-rays. To generate survival profiles in function of the repetition rate of LDP
bunches, HCT116 cells were exposed to series of 5 LDP bunches and glioblastoma cells, SF763 and U87-MG, to
series of 9 bunches. After exposure to ionizing radiations, cells were incubated for 3 hours in standard conditions.
Cells were harvested with Accutase (Merck), dispatched into 3 different wells of 12-well plates (TPP) in 2.5 ml
of medium and grown for five generations corresponding to 5 days for HCT116 and SF763 cells, and 6 days for
U87-MG cell line. Cells were harvested with Accutase which then be inactivated with the same volume of 1X PBS
(ThermoFisher Scientific) supplemented with 10% fetal calf serum. The final volume was adjusted to 1 ml with 1X
PBS and 200 µl of each well were dispatched into a non-sterile U-bottom 96-well plate (TPP). In each well, 2 µl of
a propidium iodide solution (Sigma, 100 µg/ml in 1X PBS) were added just before flow cytometry counting. Cell
®
acquisition and data analysis were performed using Guava and GuavaSoft (Merck).

DNA damage foci immunofluorescent staining. For DNA damage foci analysis, 4 × 105 cells were
®
seeded on the entire lumox surface of the dish for LDP irradiation or into Slide Flasks (NUNC) for CAP or
X-rays irradiations. Cells were subjected to 3 or 6 consecutive bunches of LDP or to 2 or 4 Gy of CAP or X-rays.
The cells were incubated one hour post irradiation at 37 °C, then washed with 1X PBS and fixed in 4% Formalin
solution (Sigma) for 15 min at room temperature. The cells were then washed twice with 1X PBS before incuba-
tion for 10 min in 1 ml of permeabilization buffer (0.5% Triton X-100 in 1X PBS). After a 20-min saturation in
®
1 ml of 1X PBS supplemented with 2% SVF, saturation buffer was entirely removed. Opened Lumox dishes were
placed 5 min under chemical hood to eliminate liquid excess. The Immunostaining surface was delimitated using
Dako Pen (Agilent). The cells were incubated for one hour at room temperature with a mouse monoclonal anti-
body against γH2AX (Merck) diluted in PBS/SVF (1/1000, 30 µl per area). The cells were then washed twice with
PBS/SVF before incubation in a 50 µl dilution of secondary antibody (Alexafluor 488 goat anti-mouse antibody,
ThermoFisher Scientific, 1/500 in PBS/SVF). After a wash with 1X PBS, the cells were incubated in 1X PBS supple-
mented with DAPI (ThermoFisher Scientific, 0.1 µg/ml) for 10 min at room temperature and were finally washed
once again with 1X PBS. Liquid excess was evaporated 5 min under chemical hood. The irradiated Lumox area ®
Scientific Reports | (2019) 9:10132 | https://doi.org/10.1038/s41598-019-46512-1 8
www.nature.com/scientificreports/ www.nature.com/scientificreports

was cut following Dako Pen delimitation and was mounted on glass microscopy coverslips. Coverslips were then
®
mounted on slides from Slide Flasks using Prolong Gold Antifade (ThermoFisher Scientific). Picture Acquisition
was performed using DMi8 microscope and LASX software (Leica) at 40x magnification.

Automated counting of DNA damage foci. Foci were automatically counted in each nucleus, using
an ImageJ macro as previously described35. Nuclear images were processed to correct eventual uneven illumi-
nation by dividing the original nuclear image by a duplicate convolved by a gaussian filter. The resulting image
intensities were enhanced and smoothed using successively a gamma transform and a median filter. Automated
threshold cut was applied, followed by morpho-mathematics operations (close, dilate, fill holes and watershed).
The “analyze particle” function was used to isolate individual nuclei and retrieve their contours. The outline of
each nucleus was drawn onto the image of DNA repair foci. Foci were characterized as local maxima, and the
corresponding ImageJ function was used to identify and count them.

Protein extraction and Western Blotting. Cells were exposed to Olaparib and/or to H2O2, medium
was removed and cells were harvested and lysed by adding SDS-Urea buffer (62.5 mM TrisHCl pH6.8, 6 mM
Urea, 10% Glycerol and 2% SDS) on ice. Cell extracts were sonicated 10 s and protein concentration deter-
mined using DCTM Protein Assay (Bio-Rad). Ten micrograms of proteins were loaded and run on NuPAGE
3–8% Tris-Acetate (ThermoFisher Scientific) before being transferred onto 0.45 µm PVDF membrane (Merck).
Immunostaining was performed by incubating membrane 1 h at room temperature with 1/1000 diluted of rabbit
polyclonal anti-Poly-ADP Ribose (TREVIGEN 4336-BPC-100), rabbit monoclonal anti-PARP1 (Cell Signaling
Technology CST#9532) or mouse monoclonal anti-GAPDH (Merck, MAB374) antibodies. Goat anti-rabbit and
goat anti-mouse IgG antiserums conjugated to peroxidase (SouthernBiotech, 4050-05 and 1031-05 respectively,
1/5000) to reveal protein signals. Images were acquired using G:BOX (Syngene).

Data analysis. Data analyses were performed using the GraphPad Prism software. The linear quadratic
model was applied to fit the survival curves. Depending on experimental design, ratio paired t-test or the two-way
ANOVA Tukey’s multiple comparisons tests, with α = 0.05, were applied: “*” corresponds to p < 0.05, “**” to
p < 0.001, “***” to p < 0.001 and “****” to p < 0.0001.

Data Availability
The datasets generated and/or analyzed during the current study are available from the corresponding author on
reasonable request.

References
1. Doyen, J., Falk, A. T., Floquet, V., Herault, J. & Hannoun-Levi, J. M. Proton beams in cancer treatments: Clinical outcomes and
dosimetric comparisons with photon therapy. Cancer treatment reviews 43, 104–112, https://doi.org/10.1016/j.ctrv.2015.12.007
(2016).
2. Durante, M. & Loeffler, J. S. Charged particles in radiation oncology. Nature reviews. Clinical oncology 7, 37–43, https://doi.
org/10.1038/nrclinonc.2009.183 (2010).
3. Bin, J. et al. A laser-driven nanosecond proton source for radiobiological studies. Applied Physics Letters 101, 243701, https://doi.
org/10.1063/1.4769372 (2012).
4. Doria, D. et al. Biological effectiveness on live cells of laser driven protons at dose rates exceeding 109 Gy/s. AIP Advances 2, 011209,
https://doi.org/10.1063/1.3699063 (2012).
5. Pommarel, L. et al. Spectral and spatial shaping of a laser-produced ion beam for radiation-biology experiments. Physical Review
Accelerators and Beams 20, 032801 (2017).
6. Tommasino, F. & Durante, M. Proton Radiobiology. Cancers 7, 353 (2015).
7. Fiorini, F. et al. Dosimetry and spectral analysis of a radiobiological experiment using laser-driven proton beams. Physics in Medicine
& Biology 56, 6969 (2011).
8. Kraft, S. D. et al. Dose-dependent biological damage of tumour cells by laser-accelerated proton beams. New Journal of Physics 12,
101088/1367-2630/12/8/085003 (2010).
9. Manti, L. et al. The radiobiology of laser-driven particle beams: focus on sub-lethal responses of normal human cells. Journal of
Instrumentation 12, C03084 (2017).
10. Raschke, S. et al. Ultra-short laser-accelerated proton pulses have similar DNA-damaging effectiveness but produce less immediate
nitroxidative stress than conventional proton beams. Scientific reports 6, 32441, https://doi.org/10.1038/srep32441 (2016).
11. Yogo, A. et al. Measurement of relative biological effectiveness of protons in human cancer cells using a laser-driven
quasimonoenergetic proton beamline. Applied Physics Letters 98, 053701, https://doi.org/10.1063/1.3551623 (2011).
12. Yogo, A. et al. Application of laser-accelerated protons to the demonstration of DNA double-strand breaks in human cancer cells.
Applied Physics Letters 94, 181502, https://doi.org/10.1063/1.3126452 (2009).
13. Zeil, K. et al. Dose-controlled irradiation of cancer cells with laser-accelerated proton pulses. Applied Physics B 110, 437–444, https://
doi.org/10.1007/s00340-012-5275-3 (2013).
14. Ponette, V. et al. Hyperfast, early cell response to ionizing radiation. International journal of radiation biology 76, 1233–1243 (2000).
15. Fernet, M. et al. Poly(ADP-ribose) polymerase, a major determinant of early cell response to ionizing radiation. International journal
of radiation biology 76, 1621–1629 (2000).
16. Langelier, M. F. & Pascal, J. M. PARP-1 mechanism for coupling DNA damage detection to poly(ADP-ribose) synthesis. Current
opinion in structural biology 23, 134–143, https://doi.org/10.1016/j.sbi.2013.01.003 (2013).
17. Chen, A. PARP inhibitors: its role in treatment of cancer. Chinese journal of cancer 30, 463–471, https://doi.org/10.5732/
cjc.011.10111 (2011).
18. de Murcia, J. M. et al. Requirement of poly(ADP-ribose) polymerase in recovery from DNA damage in mice and in cells. Proceedings
of the National Academy of Sciences 94, 7303–7307, https://doi.org/10.1073/pnas.94.14.7303 (1997).
19. Nile, D. L., Rae, C., Hyndman, I. J., Gaze, M. N. & Mairs, R. J. An evaluation in vitro of PARP-1 inhibitors, rucaparib and olaparib, as
radiosensitisers for the treatment of neuroblastoma. BMC cancer 16, 621, https://doi.org/10.1186/s12885-016-2656-8 (2016).
20. Ray Chaudhuri, A. & Nussenzweig, A. The multifaceted roles of PARP1 in DNA repair and chromatin remodelling. Nature reviews.
Molecular cell biology 18, 610–621, https://doi.org/10.1038/nrm.2017.53 (2017).
21. Wilks, S. C. et al. Energetic proton generation in ultra-intense laser–solid interactions. Physics of Plasmas 8, 542–549, https://doi.
org/10.1063/1.1333697 (2001).

Scientific Reports | (2019) 9:10132 | https://doi.org/10.1038/s41598-019-46512-1 9

www.nature.com/scientificreports/ www.nature.com/scientificreports

22. Bulanov, S. V. & Khoroshkov, V. S. Feasibility of using laser ion accelerators in proton therapy. Plasma Physics Reports 28, 453–456,
https://doi.org/10.1134/1.1478534 (2002).
23. Malka, V. et al. Practicability of protontherapy using compact laser systems. Medical Physics 31, 1587–1592, https://doi.
org/10.1118/1.1747751 (2004).
24. Masood, U. et al. A light-weight compact proton gantry design with a novel dose delivery system for broad-energetic laser-
accelerated beams. Physics in medicine and biology 62, 5531–5555, https://doi.org/10.1088/1361-6560/aa7124 (2017).
25. Ponette, V. et al. Pulse exposure to ionizing radiation elicits rapid changes in cellular radiosensitivity. J Comptes rendus de l’Academie
des sciences. Serie III, Sciences de la vie 319(6), 505–509 (1996).
26. Wei, H. & Yu, X. Functions of PARylation in DNA Damage Repair Pathways. Genomics, Proteomics & Bioinformatics 14, 131–139,
https://doi.org/10.1016/j.gpb.2016.05.001 (2016).
27. https://clinicaltrials.gov.
28. Dungey, F. A., Löser, D. A. & Chalmers, A. J. Replication-Dependent Radiosensitization of Human Glioma Cells by Inhibition of
Poly(ADP-Ribose) Polymerase: Mechanisms and Therapeutic Potential. International Journal of Radiation Oncology • Biology •
Physics 72, 1188–1197, https://doi.org/10.1016/j.ijrobp.2008.07.031 (2008).
29. Shervington, A. et al. Telomerase subunits expression variation between biopsy samples and cell lines derived from malignant
glioma. Brain Research 1134, 45–52, https://doi.org/10.1016/j.brainres.2006.11.093 (2007).
30. Makvandi, M. et al. A PET imaging agent for evaluating PARP-1 expression in ovarian cancer. The Journal of Clinical Investigation
128, 2116–2126, https://doi.org/10.1172/JCI97992 (2018).
31. Li, B., Navarro, S., Kasahara, N. & Comai, L. Identification and biochemical characterization of a Werner’s syndrome protein
complex with Ku70/80 and poly(ADP-ribose) polymerase-1. The Journal of biological chemistry 279, 13659–13667, https://doi.
org/10.1074/jbc.M311606200 (2004).
32. Haince, J. F. et al. PARP1-dependent kinetics of recruitment of MRE11 and NBS1 proteins to multiple DNA damage sites. The
Journal of biological chemistry 283, 1197–1208, https://doi.org/10.1074/jbc.M706734200 (2008).
33. Bitler, B. G., Watson, Z. L., Wheeler, L. J. & Behbakht, K. PARP inhibitors: Clinical utility and possibilities of overcoming resistance.
Gynecologic Oncology 147, 695–704, https://doi.org/10.1016/j.ygyno.2017.10.003 (2017).
34. Zhou, J. X., Feng, L. J. & Zhang, X. Risk of severe hematologic toxicities in cancer patients treated with PARP inhibitors: a meta-
analysis of randomized controlled trials. Drug design, development and therapy 11, 3009–3017, https://doi.org/10.2147/DDDT.
S147726 (2017).
35. Bayart, E. et al. Enhancement of IUdR Radiosensitization by Low-Energy Photons Results from Increased and Persistent DNA
Damage. PloS one 12, e0168395, https://doi.org/10.1371/journal.pone.0168395 (2017).
36. Calugaru, V. et al. Radiobiological Characterization of Two Therapeutic Proton Beams With Different Initial Energy Spectra Used
at the Institut Curie Proton Therapy Center in Orsay. International Journal of Radiation Oncology • Biology • Physics 81, 1136–1143,
https://doi.org/10.1016/j.ijrobp.2010.09.003 (2011).
37. Schillaci, F. et al. Characterization of the ELIMED Permanent Magnets Quadrupole system prototype with laser-driven proton beams,
Vol. 11 (2016).

Acknowledgements
The authors acknowledge the support of OSEO Project No. I0901001W-SAPHIR and of the European Research
Council through the X-Five ERC project (Contract No. 339128). F.M.C. aknowledges funding from “France
Hadrons” for the CAP irradiation facilities. E. B. acknowledges support from the European Union’s Horizon 2020
research and innovation program (H2020-INFRAIA-2014-2015) under Grant Agreement No. 654148 Laserlab-
Europe. This work was also supported by Paris-Saclay University IRS financing, NanoTherad project and EDF
Grant No. RB 2017-21 and RB 2018-15.

Author Contributions
The study was planned and supervised by E.B., A.F., F.M.C., E.D. and V.M. Experimental setup and data
acquisition was performed by E.B., O.D., L.P., D.L. and M.C. with help by F.M.C. and A.F. Data evaluation and
writing of the publication manuscript was jointly done by E.B., F.M.C. and A.F. with help by E.D. and V.M.

Additional Information
Competing Interests: The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre-
ative Commons license, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons license and your intended use is not per-
mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the
copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

© The Author(s) 2019

Scientific Reports | (2019) 9:10132 | https://doi.org/10.1038/s41598-019-46512-1 10