ORIGINAL RESEARCH

published: 29 July 2021

doi: 10.3389/fonc.2021.686142

Cancer Cells Can Exhibit a Sparing

FLASH Effect at Low Doses Under
Normoxic In Vitro-Conditions
Gabriel Adrian 1,2*, Elise Konradsson 3, Sarah Beyer 1, Anders Wittrup 1,2,4,
Karl T. Butterworth 5, Stephen J. McMahon 5, Mihaela Ghita 5, Kristoffer Petersson 2,6
and Crister Ceberg 3
1 Division of Oncology and Pathology, Clinical Sciences, Skåne University Hospital, Lund University, Lund, Sweden,
2 Department of Hematology, Oncology and Radiation Physics, Skåne University Hospital, Lund, Sweden, 3 Department of
Medical Radiation Physics, Clinical Sciences, Lund University, Lund, Sweden, 4 Wallenberg Center for Molecular Medicine,
Lund, Sweden, 5 Patrick G. Johnston Centre for Cancer Research, Queen’s University Belfast, Belfast, United Kingdom,
6 Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford,

United Kingdom
Edited by:
Lorenzo Manti,
University of Naples Federico II, Italy
Background: Irradiation with ultra-high dose rate (FLASH) has been shown to spare
Reviewed by:
normal tissue without hampering tumor control in several in vivo studies. Few cell lines
Pavel Blaha,
National Institute of Nuclear Physics of have been investigated in vitro, and previous results are inconsistent. Assuming that
Naples, Italy oxygen depletion accounts for the FLASH sparing effect, no sparing should appear for
Martina C. Fuss,
GSI Helmholtz Center for Heavy Ion
cells irradiated with low doses in normoxia.
Research, Germany Methods: Seven cancer cell lines (MDA-MB-231, MCF7, WiDr, LU-HNSCC4, HeLa [early
*Correspondence: passage and subclone]) and normal lung fibroblasts (MRC-5) were irradiated with doses
Gabriel Adrian
gabriel.adrian@med.lu.se ranging from 0 to 12 Gy using FLASH (≥800 Gy/s) or conventional dose rates (CONV, 14
Gy/min), with a 10 MeV electron beam from a clinical linear accelerator. Surviving fraction
Specialty section: (SF) was determined with clonogenic assays. Three cell lines were further studied for
This article was submitted to
Radiation Oncology,
radiation-induced DNA-damage foci using a 53BP1-marker and for cell cycle
a section of the journal synchronization after irradiation.
Frontiers in Oncology
Results: A tendency of increased survival following FLASH compared with CONV was
Received: 26 March 2021
Accepted: 07 July 2021 suggested for all cell lines, with significant differences for 4/7 cell lines. The magnitude of
Published: 29 July 2021 the FLASH-sparing expressed as a dose-modifying factor at SF=0.1 was around 1.1 for 6/
Citation: 7 cell lines and around 1.3 for the HeLasubclone. Similar cell cycle distributions and 53BP1-
Adrian G, Konradsson E, Beyer S,
Wittrup A, Butterworth KT,
foci numbers were found comparing FLASH to CONV.
McMahon SJ, Ghita M, Conclusion: We have found a FLASH effect appearing at low doses under normoxic
Petersson K and Ceberg C (2021)
Cancer Cells Can Exhibit a Sparing conditions for several cell lines in vitro. The magnitude of the FLASH effect differed
FLASH Effect at Low Doses Under between the cell lines, suggesting inherited biological susceptibilities for FLASH irradiation.
Normoxic In Vitro-Conditions.
Front. Oncol. 11:686142. Keywords: FLASH, ultra-high dose rate irradiation, clonogenic assay, normoxia, radiotherapy, radiobiology,
doi: 10.3389/fonc.2021.686142 radioresistance, cancer cell lines

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Adrian et al. Clonogenic FLASH Sparing in Normoxia

INTRODUCTION MATERIAL AND METHODS

The FLASH effect denotes the radiobiological phenomenon that Cell Culture
a given absorbed dose of ionizing radiation produces less damage The human breast cancer cell lines MCF7 and MDA-MB-231,
at ultra-high dose rates (>40-100 Gy/s), as compared to the lower the human fibroblast cell line MRC-5, and the human cervix
dose rates conventionally used in radiotherapy (CONV, about cancer cell line HeLa (in the study two different HeLa cells were
0.1 Gy/s). Experimental evidence for the FLASH effect has been used; early passage cells and a high passage subclone) were
demonstrated in vivo in various preclinical settings (1–5), as well acquired from American Type Culture Collection (ATCC).
as in one first-in-human case of a patient with multi-resistant The human colon cancer cell line WiDr was acquired from
cutaneous lymphoma (6). LGC Promochem (Teddington, UK). The squamous cell
Although there is no proven mechanistic explanation for the carcinoma LU-HNSCC4 was established in our laboratory
FLASH effect, the limited in vivo data available suggest that more from a patient with a squamous cell carcinoma in the floor of
sparing may occur in physoxic normal tissues than in severely the mouth (21). Cells were grown in monolayers in DMEM
hypoxic or nearly anoxic tumors (7). One plausible theory (MCF7, MDA-MB-231, WiDr, HeLa, LU-HNSCC4) or EMEM
describes the FLASH effect as a protective, radiation-induced (MRC5) media with 10% fetal bovine serum and 1% penicillin-
hypoxia, tentatively explained by the so-called transient oxygen streptomycin at 37°C in a humidified atmosphere with 5%
depletion (TOD) hypothesis as a net effect of radiolytic oxygen CO 2 . All cell lines were confirmed to be negative for
consumption exceeding the physiologic supply (8–10). mycoplasma infection.
According to the TOD hypothesis, the degree of sparing would
be largest for already hypoxic tissues, where further oxygen Clonogenic Assays
depletion can be substantial. No effect would be expected at Exponentially growing cells were trypsinized and plated in
normoxia, where radiolytic oxygen consumption would not be appropriate cell densities in 2.50 ml medium per Falcon T12.5
sufficient for producing hypoxic radioresistance, or at anoxia flask (Thermo Fischer Scientific TM, Waltham, MA) and allowed
where there can be no further oxygen depletion. However, recent to adhere overnight before irradiation. Control flasks for
work by Labarbe et al. has indicated, based on simulations and determination of the plating efficiency and the FLASH- and
mathematical modelling, that the TOD hypothesis is most likely CONV-flasks were prepared identically on the same occasion,
not sufficient to account for the FLASH effect reported at dose for each repetition. FLASH and CONV-flasks were irradiated
levels limited by normal tissue toxicity (11). Consequently, the minutes apart with doses from 0-9 Gy (12 Gy for HeLasubclone).
authors suggest that other mechanisms may regulate the process Irradiation was performed under normoxic conditions at room
and that a FLASH effect may be present also at normoxic temperature with the flasks lying flat and irradiated from beneath
conditions, even at relatively low dose levels. (beam angle 180 degrees). After irradiation, the flasks were
Surprisingly, few recent in vitro studies with clonogenic assays returned to the incubator for 9-14 days. All flasks, including
have been reported to support these basic assumptions, and the the non-irradiated controls, were terminated at the same
limited data available suggests that different cell lines may have occasion. Cells were fixed and stained with methylene-blue in
different susceptibility to the FLASH effect. In previous work, we 70% ethanol. Flasks were scanned using a flatbed scanner in
studied clonogenic survival of the human prostate cancer DU145 1,200 dpi resolution. Colony counts were performed with a
cell line and found a FLASH effect at lower oxygen standardized ImageJ-code (version 1.53e, Wayne Rasband,
concentrations but no significant differences in normoxic National Institute of Health, USA) and manually checked.
conditions (12). Montay-Gruel et al. studied the murine Surviving fraction (SF) was determined as the number of
glioblastoma H454 cell line and demonstrated significant colonies with at least 50 cells divided by the number of plated
FLASH effects both at 4% oxygen concentration and in cells (corrected for plating efficiency).
normoxic conditions (13). For normal human lung fibroblasts
(14) and lung cancer A549 (15), no difference in survival at DNA-Double Strand Break Foci Formation
different dose-rates in normoxia was reported. Neither has the 150 000 - 500 000 cells were plated in Slide-Flasks (Thermo
use of laser-accelerated protons revealed any dose-rate Fischer Scientific Nunc, Roskilde, Denmark) and allowed to
dependent differences in normoxia (16–18). Contrary to these adhere overnight before irradiation with 3 Gy. At specific time
findings, for two murine pancreatic cancer cell lines, Venkatesulu points after irradiation, cells were washed with PBS and fixed
et al. found a reversed FLASH effect at normoxic conditions (19). with 4% paraformaldehyde for 20 min. After washing, cells were
Consequently, there is a need for further in vitro studies permeabilized with 0.5% Triton-X100 in PBS for 20 min, washed,
allowing for experiments in a controlled oxygen environment blocked in blocking buffer (0.2% skimmed milk, 0.1% TritonX-
(1, 20). In the present work, we have performed a comparative 100, 5% FBS in PBS) for 1 hour followed by 1 hour incubation
study of FLASH vs. CONV and assessed clonogenic survival, with 53BP1 primary antibody (Invitrogen PA146147) and 1 hour
DNA damage, and cell cycle synchronization under normoxic of incubation with a secondary antibody (AlexaFluor anti-rabbit
conditions for a range of different cell lines. Our investigations 488). C ell nuc lei were co unt erstain ed with D API.
show that the FLASH effect may occur at relatively low doses Permeabilization, washing, blocking and staining steps were all
under normoxic conditions and that it depends on cell-line performed at room temperature. Foci formation were assessed
specific variations in susceptibility. with a widefield fluorescence microscope, AxioOberver Z.1

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Adrian et al. Clonogenic FLASH Sparing in Normoxia

(Zeiss, Oberkochen, Germany), equipped with ×63/1.40 Plan- common a and b parameters for all data. The residuals were
Apochromat oil-immersion objective lens and Colibri 7 solid tested for normality using the Kolmogorov-Smirnov test, and the
state LED light source (Zeiss), and an ORCA-Flash4.0 V3 Digital F-test was used to determine whether the fit was significantly
CMOS camera (Hamamatsu Photonics, Hamamatsu City, improved by using separate parameters. Using the model fitted
Japan). In each sample, at four different positions, fifteen Z- with separate a and b parameters for the CONV and FLASH
stack images were acquired, deconvoluted with a GPU-based data, dose-modifying factors (DMF) were determined as the ratio
deconvolution module and averaged using a Maximum Intensity of DFLASH/DCONV at a survival fraction of 0.1 (SF=0.1) and 0.01
Projection-algorithm (Black Zen Imaging Software, Zeiss). (SF=0.01). Boot-strapping was used to calculate the median and
ImageJ was used for automated foci identification and the interquartile range of the DMF. In addition, the difference in
quantification of DNA-double strand break (DSB) foci. survival fraction at the individual dose levels were tested for
statistical significance, without assuming normality, by using the
Cell Cycle Analyses Wilcoxon rank-sum test. All tests were two-sided with a chosen
150 000 - 500 000 cells were plated in 35 mm or 60 mm Petri significance level of 5%. Experiments were repeated 2-4 times.
Dishes (Corning, Corning, NY, USA) and allowed to adhere
overnight before irradiation with 6 Gy (and 3 Gy for the
HeLasubclone cells). At 24 h (6 h and 24 h for HeLasubclone cells),
after irradiation cells were washed with PBS, harvested and RESULTS
fixated with ice-cold ethanol (70%). Cell nuclei were stained
with propidium iodide (10 lg/ml, RNase A 0.1 lg/ml) for 30 min at Clonogenic Assays
room temperature and DNA content was determined with an A tendency of increased survival after FLASH compared with
Accuri C6 Flow Cytometer (Becton Dickinson, Franklin Lakes, CONV was suggested for all cell lines (Figure 1), with significant
NJ, USA). DNA-histograms were analyzed in ModFit LT 5.0 for differences for four of the seven cell lines. A general FLASH-
Mac (BD Biosciences). sparing was also indicated by a DMF at SF=0.1 of around 1.1 for
all cell lines, except the HeLasubclone, for which it was around 1.3
Irradiation and Dosimetry (Table 1). The use of separate a and b parameters for FLASH
Irradiation and dosimetry were performed as described and CONV resulted in significantly improved fits for the MCF7,
previously (12). In summary, a modified (22) Elekta Precise LU-HNSCC4, HeLaearly passage and HeLasubclone, indicating
(Elekta AB, Stockholm, Sweden) medical linear accelerator differences across the curves as a whole. Significant survival
(LINAC) was used for irradiations with FLASH and CONV differences were also observed for MDA-MB-231 at 6 Gy and 9
dose rates with a 10 MeV electron beam. The average dose rate Gy. The WiDr and MRC-5 cell lines did not show any significant
for CONV irradiation was 14 Gy/min. For FLASH irradiation, differences in survival after FLASH compared with CONV in the
the average dose rate was ≥800 Gy/s, delivered with an integer dose range studied.
number of 3.5 ms pulses, with a dose-per-pulse of 3.0 Gy, and a
pulse repetition frequency of 200 Hz. Thus, the instantaneous DSB—Foci With 53BP1
pulse dose rate was 0.86 MGy/s, which is the same as the average Three of the cancer cell lines, LU-HNSCC4, MDA-MB-231 and
dose rate for the 3 Gy single pulse delivery, while the average HeLasubclone, were further studied for radiation-induced DNA-
dose rate was 1.2 kGy/s for the 6 Gy delivery, 900 Gy/s for the 9 DSB using the 53BP1-marker at 2 h and 24 h after irradiation
Gy delivery, and 800 Gy/s for the 12 Gy delivery. GafChromic with 3 Gy (Figure 2). A marked induction of DNA-DSB foci was
EBT3 film (Ashland Specialty Ingredients G.P., Bridgewater, NJ) seen at 2 h after irradiation, and declined substantially at 24 h.
was used for dosimetry for both FLASH and CONV irradiation. Comparing FLASH and CONV, median foci numbers were
Dose measurements were performed in conjunction with each similar with overlapping interquartile ranges, for all three
cell experiment. In addition, online dose delivery verification studied cell lines (Table 2).
measurements were performed. For CONV irradiation, these
were performed with the built-in monitor (transmission) Cell Cycle Analyses
chamber. For FLASH irradiation, a Farmer-type ionization To further investigate potential differences in radiation response
chamber placed at a specific position in the ceiling of the between FLASH and CONV, radiation-induced cell cycle arrest was
treatment room (furthest possible distance from the source) investigated for MDA-MD-231, LU-HNSCC4 and HeLasubclone
was used. cells. At 24 hours after irradiation with 6 Gy, both FLASH and
CONV induced cell cycle synchronizations in the three cell lines
Statistical Analyses (Figures 3A–C). Interestingly, HeLasubclone cells were predominantly
RStudio v. 1.0.136 (RStudio Team (2015). RStudio, Inc., Boston, synchronized in early S-phase (the S-phase was sub-analyzed in
MA, URL http://www.rstudio.com/) was used for statistical three compartments, Supplementary Figure S1), whereas the
calculations. The parameters of the linear–quadratic model MDA-MB-231 and LU-HNSCC4 cells were synchronized in the
(23) (SF=exp(−aD−bD2)) were fitted to the log (SF) using the G2/M-phase. To elucidate if the HeLasubclone cell synchronization in
nonlinear least-squares method (‘nls’ in RStudio). Two early S-phase was due to a previous transient G2/M-arrest, we
alternative models were fitted, one with separate a and b studied an earlier time point, 6 h after irradiation and an additional
parameters for the CONV and FLASH data, and one with dose level, 3 Gy, and found radiation-induced G2/M-arrest

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Adrian et al. Clonogenic FLASH Sparing in Normoxia

A B

C D

E F

FIGURE 1 | Surviving fraction assessed by clonogenic assay comparing FLASH with conventional dose rates (CONV) for human in vitro-cell lines; (A) Breast cancer
cell line MDA-MB-231, (B) Breast cancer cell line MCF7, (C) Cervix cancer cell line HeLaearly passage, (D) HeLasubclone (E) Head&neck cancer cell line LU-HNSCC4,
(F) Colon cancer cell line WiDr, and (G) Normal lung fibroblasts MRC-5. Blue circles denote FLASH, red squares denote CONV, and grey circles denote the non-
irradiated controls. The empty symbols represent the individual flasks and the filled symbols represent the average surviving fraction at the dose indicated. The
dashed lines illustrate the fitted survival curve according to the linear quadratic model. Diamond symbols denote samples below the detection limit (no surviving
colonies). Statistical analyses using Wilcoxon Rank-Sum test; ns, not significant, *p < 0.05, **p < 0.01. Data from three independent experiments.

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Adrian et al. Clonogenic FLASH Sparing in Normoxia

TABLE 1 | Dose modifying factors (DMF) at a surviving fraction (SF) of 0.1 and 0.01 for the various cell lines.

Cell line DMF (SF=0.1) IQR (SF=0.1) DMF (SF=0.01) IQR (SF=0.01) F-test p-value

WiDr 1.16 1.03-1.29 1.20 1.10-1.30 0.34

MCF7 1.10 1.00-1.20 1.16 1.11-1.21 0.03
LU-HNSCC4 1.12 0.94-1.30 1.15 1.06-1.24 0.007
MRC-5 1.09 0.97-1.21 1.05 1.00-1.10 0.24
MDA-MB-231 1.14 1.02-1.26 1.12 1.07-1.17 0.15
HeLaearly passage 1.12 1.02-1.22 1.13 1.09-1.17 0.04
HeLasubclone 1.32 1.19-1.45 NA NA 0.05

The F-test denotes the significance level for separate parameter sets for FLASH and CONV, compared with one common fit. IQR; inter-quartile range; NA, Not Applicable.

(Figure 3D). The cell cycle analyses could not resolve any significant with a significant sparing already apparent at 3 Gy. The normal lung
differences between FLASH and CONV. fibroblasts did not show any significant difference in survival between
FLASH and CONV. Cell cycle synchronization and DSB-foci
formation were assessed for three of the cancer cell lines with
DISCUSSION similar responses for FLASH and CONV exposures.
It is well recognized that FLASH spares normal tissues in vivo
We have found in vitro evidence of a FLASH sparing effect measured (2–4, 13, 24). However, available in vitro results with clonogenic
with clonogenic survival occurring under normoxic conditions for assays in normoxia are inconsistent both in recent (12–15, 19)
several cancer cell lines. The magnitude of the FLASH effect differed and older studies (25–30). Consistent with our current findings,
between the cell lines and was most pronounced for HeLasubclone cells, an increased survival fraction after irradiation with ultra-high

B C D

FIGURE 2 | Evaluation of radiation-induced DNA-double strand break foci using 53BP1. (A) Representative microscopy image showing (left to right) 53BP1, DAPI,
merged image, and the resulting analyzed image after processing in ImageJ. (B–D) Number of 53BP1 foci at 2 h and 24 h after 3 Gy irradiation with FLASH (blue) or
conventional dose rate (CONV, red) compared with controls (grey) for LU-HNSCC4 (B; 1,532 scored cells), MDA-MB-231 (C; 2,583 scored cells), and HeLasubclone
(D; 2,973 scored cells). The box and whisker plots illustrate median (thick line), interquartile range (box) and the lowest/highest observation within ±1.5* interquartile
range (IQR) from the box (whiskers). The individually scored cells are indicated with transparent circles. Data from two independent experiments.

TABLE 2 | Number of DSB-foci per cell with the 53BP1-marker for three cell lines at 2 h and 24 h after irradiation with FLASH or conventional dose rate (CONV), and
for non-irradiated controls (Ctrl).

Cell line Ctrl at 2 h FLASH at 2 h CONV at 2 h Ctrl at 24 h FLASH at 24 h CONV at 24 h

Median (IQR) Median (IQR) Median (IQR) Median (IQR) Median (IQR) Median (IQR)

LU-HNSCC4 6 (3–12) 25 (20–31) 26 (22-33) 4 (2-8) 10 (6-16) 10 (6-16)

MDA-MB-231 2 (1-5) 26 (22-31) 25 (20-30) 2 (1-4) 6 (3-9) 6 (4-10)
HeLasubclone 4 (2-9) 32 (25-40) 30 (24-37) 3 (1-6) 5 (3-8) 5 (2-9)

IQR, Inter-quartile range.

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Adrian et al. Clonogenic FLASH Sparing in Normoxia

A B

C D

FIGURE 3 | Cell cycle distributions determined by flow cytometry after irradiation with FLASH or conventional dose rate (CONV). In (A–C), cell cycle distribution 24 h
after irradiation with 6 Gy for LU-HNSCC4 (A), MDA-MB-231 (B) and HeLasubclone (C). In (D) an earlier time-point (6 h) after irradiation with 3 Gy and 6 Gy using the
HeLasubclone. Bars illustrate G1 (light grey), S-phase (grey), and G2/M (black). The figures in the bars denote the percentage of cells (with standard deviations). Data
from two independent experiments.

dose rate in normoxia have been reported (13, 29, 30), whereas The present results showing differences between cell lines,
other results indicate no difference (14, 15, 25–28) or a reversed together with the inconsistent findings by others, suggest that
effect (19). The inconsistent results could indicate differing the FLASH effect might not be an independent, universal dose-
intrinsic biological susceptibility for FLASH. In addition, modifying factor. Instead, the sparing effect could involve
survival differences have been shown for hypoxic in vitro- biological determinants varying from cell line to cell line.
conditions (12, 27, 28, 31, 32). In the present study, the DSB-foci formations were numerically in the same range for
magnitude of the FLASH effect, expressed as DMF at SF=0.1, the three studied cancer cell lines, with similar foci numbers for
was around 1.1 for six of the seven cell lines, while a DMF of 1.3 FLASH compared with CONV irradiation. Fouillade et al., using
was found for the HeLasubclone. The values are in line with the same DSB-marker at an earlier time point (30 minutes),
previously published in vivo data, generally showing a DMF of showed a lower number of foci for FLASH compared with
1.2-1.5 (7). The HeLasubclone data (with passage number CONV for normal lung fibroblasts, but no differences for the
approaching 40) show a distinct behavior compared to the A549 lung cancer cell line (24). Cell cycle synchronization after
other cell lines, with a larger DMF and also earlier cell cycle irradiation was seen for the three studied cell lines in the current
arrest. In comparison with HeLaearly passage, the HeLasubclone was study, but with similar effects after both FLASH and CONV
considerably more radioresistant and lacked a shouldered irradiation. Auer et al. also studied cell cycle synchronization of
survival curve for the CONV-irradiated samples. It has HeLa cells after irradiation with 3 Gy using laser-accelerated
previously been reported that phenotype changes can occur protons at different dose rates. They found a less pronounced
with high passage numbers, affecting radiation responses (33). G2/M-accumulation at 10 h for cells irradiated with ultra-high

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Adrian et al. Clonogenic FLASH Sparing in Normoxia

dose rate compared with conventional dose rate, but no interactions was minimized. Thereby, the results are valid under
differences at 24 h (34). The HeLasubclone cells used in the these circumstances, and their generalizability to more complex
current study revealed no synchronization in G2/M-phase at biological systems need to be further investigated. The lack of a
24 h after irradiation, instead the cells were synchronized in early difference in the DSB-foci induction between FLASH and CONV
S-phase. Additional experiments at an earlier time-point (6 h warrants future experiments using additional methods to assess
after irradiation) with two different doses (3 Gy and 6 Gy) DNA-damage and repair after irradiation. The varying results
indicated an earlier radiation induced G2/M-synchronization, between cell lines suggest that biological determinants may affect
suggesting a peak of the G2/M-arrest at a time point before 24 h the response, but this study alone does not identify any
(Supplementary Figure S1). However, the cell cycle underlying mechanisms or predictive signatures that could be
synchronization was similar after FLASH and CONV at both further examined in a preclinical or clinical translation.
6 h and 24 h. To conclude, we have found a FLASH effect under normoxic
FLASH effects are typically seen at doses ≥ 10 Gy. The current conditions for several cell lines in vitro, and that the magnitude
study showed a separation of the survival curves at doses below of the FLASH effect differed between the cell lines. The results
10 Gy. Interestingly, also using a low dose of 4 Gy, Chabi et al. indicate that the FLASH effect cannot be solely explained by
found FLASH irradiation to be more efficient than conventional TOD and that other mechanisms are involved. The nature of
dose rate exposures for two cases of T-cell lymphoblastic such possible biological susceptibilities and their dependence on
leukemia (T-ALL), but an opposite relation for a third case oxygen concentrations will be subject to further investigations.
(35). The results underpin that FLASH effects do not exclusively
occur at high doses and also suggest that intrinsic biological
factors might determine the FLASH response. The authors
proposed that genomic profiles might predict when FLASH is
DATA AVAILABILITY STATEMENT
beneficial. Additional investigations in the nature and The datasets presented in this article are not readily available but
mechanism of such biological determinants, and their the datasets generated during and/or analyzed during the current
influence on the radiochemical and biological steps of the study are available from the corresponding author on reasonable
radiation response remain to be investigated. Many of the steps request. Requests to access the datasets should be directed to
are likely to be influenced by the available oxygen concentration, gabriel.adrian@med.lu.se.
and we have previously shown the dependence on oxygen
concentration for a FLASH effect (12). However, differences
between FLASH and CONV at low doses in well-oxygenated
environments, i.e. where oxygen depletion is considered to be AUTHOR CONTRIBUTIONS
negligible, imply that the TOD hypothesis is insufficient to
GA, CC, SM, and KP designed the study. GA, SB, EK, KB, MG,
account for the whole FLASH effect. We therefore deduce that
and KP conducted the experiments. GA, CC, EK, SM, SB, KP,
the FLASH effect, in part, must be caused by other mechanisms.
and AW analyzed and interpreted the data GA, KP, and CC
FLASH radiotherapy is a promising new technique and
drafted the manuscript. Critical revision of the manuscript for
convincing reports show its ability to protect normal tissue
important intellectual content – all authors. GA and CC did the
from radiation damage (2–5, 13, 24). Most in vivo experiments
statistical analyses. All authors contributed to the article and
also suggest an iso-effective tumor control compared with
approved the submitted version.
CONV (2, 5, 36–38), even though some studies have found
other results (19, 35). The tumor’s response to radiation in the
complex in vivo environment is dependent not only on direct cell
kill but also on inflammatory reactions and the immune system, FUNDING
involving surrounding connective tissues. Considering a possible
inherited susceptibility for cancer cells to exhibit a FLASH effect This work was supported by Mrs Berta Kamprad Foundation
(35), which would be detrimental for tumor control, further (grant no 2020-19-301), Swedish Cancer Society (grant no 20
investigation in the differential response between tumor and 1298), John and Augusta Persson’s Foundation and
normal tissue is clearly indicated. Ideally, such studies will governmental research funding (ST-ALF). Funding for AW
generate models that describe tissues and tumors for which a was p rovided by the Swedish Socie ty for Medical
differential response can be exploited. Research (SSMF).
The current study has some limitations. Since the FLASH
irradiation was delivered with an integer number of pulses (1-4),
the average dose rate varied between the different dose levels. ACKNOWLEDGMENTS
However, the dose-per-pulse and the instantaneous/pulse dose
rate were constant and the average dose rate exceeded 800 Gy/s Parts of the results were presented at the Annual Meeting of
at all dose levels. Further, we have chosen to perform a pure in European Radiation Research Society in Lund, Sweden, in
vitro study to enable studies of different cell lines under well- September 2020. The content in the article is part of a PhD-
controlled oxygen concentrations where the impact of biological thesis by GA.

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Adrian et al. Clonogenic FLASH Sparing in Normoxia

Supplementary Figure 1 | Cell cycle distributions determined by flow cytometry

SUPPLEMENTARY MATERIAL after irradiation with FLASH or conventional dose rate (CONV) for the HeLasubclone
cell line 24 h after irradiation with 6 Gy. The S-phase was sub-divided into S1 (early),
The Supplementary Material for this article can be found online
S2 (middle) and S3 (late). Bars illustrate G1 (light grey), S-phase (grey), and G2/M
at: https://www.frontiersin.org/articles/10.3389/fonc.2021. (black). The figures in the bars denote the percentage of cells (with standard
686142/full#supplementary-material deviations). Data from two independent experiments.

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