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and 53BP1) after FLASH vs. conventional dose-rate
irradiation (CONV, 0.03 Gy/s).
Material and Methods
We used two non-transformed human lung fibroblasts,
MRC5 and IMR 90 and one human lung cancer cell line,
A549. Cells were grown on coverslips and given 5 Gy with
the same LINAC (4.5 MeV electrons) in the FLASH or CONV
mode 24 h after seeding. EBT3 Gafchromic films were used
to assess the dose received by each flask. Cell viability was
evaluated 6 days post-irradiation (pi) by a MTT assay for
the three cell lines and manual cell counting for the two
fibroblastic cell lines. A clonogenic survival assay was also
performed for A549 cells. For DNA damage response
studies, γH2AX and 53BP1 foci were detected by an
immunofluorescence method. To quantify those foci, z-
stack images across the nucleus were acquired on a SP5
Leica confocal microscope and foci analyzed using the 3D
object counter software. The data came from 2 - 4
experiments performed under the same conditions and
analysed with the same modalities. The statistical tests
were Wilcoxon (cell viability) and Student (DNA damage
response).
Results
The MTT assay did not show any difference between CONV
vs. FLASH modalities. For A549 cells, the median of the
MTT signal was 86.6% after CONV vs. 78.8% after FLASH
(p=0.7); for MRC5, 61.3% (CONV) vs. 57% (FLASH, p=0.4);
and for IMR 90, 72.7% (CONV) vs. 72.5% (FLASH, p=1). Cell
counting did not find any difference too: for MRC5 cells,
the median of the % of live cells was 20% (CONV) vs. 15.9%
(FLASH, p=0.1); for IMR 90, 15.8% (CONV) vs. 15.1%
(FLASH, p=0.3). The clonogenic survival assay did not show
any difference for A549 after CONV vs. FLASH.
For DNA damage study, the mean number of γH2AX and
53BP1 foci was determined for the three cell lines, at 30
and 180 min pi. No statistically significant difference was
observed between the two modalities of irradiation. For
A549 cells, the mean number of γH2AX foci, 30 min pi, was
30 ± 9 after CONV vs. 29 ± 10 after FLASH (p=0.6); for
MRC5, 31 ± 10 for the two modalities of irradiation; and
for IMR 90, 37 ± 11 after CONV vs. 39 ± 15 after FLASH
(p=0.36).
Conclusion
This in vitro study did not elicit any significant difference,
between CONV and FLASH, in both tumoral and non-
tumoral cell lines with cell viability and foci of DNA
damage repair proteins as endpoints. This suggests that
the differences evidenced from in vivo studies result from
the microenvironment and/or immune responses.
OC-0031 Global changes in the glycosylation of
irradiated endothelial cells with functional
consequences
C. Jaillet
1
, W. Morelle
2
, M.C. Slomianny
2
, V. Paget
1
, G.
Tarlet
1
, V. Buard
1
, S. Selbonne
1
, F. Caffin
1
, E. Rannou
1
,
P. Martinez
2
, A. François
1
, F. Foulquier
2
, F. Allain
2
, F.
Milliat
1
, O. Guipaud
1
1
Institute for Radiological Protection and Nuclear Safety,
PRP-HOM, Fontenay aux Roses, France
2
University of Lille, UGSF, Lille, France
Purpose or Objective
Altered by ionizing radiation, the vascular network is
considered as a prime target to limit normal tissue
damages and improve tumor control in the context of
radiotherapy. Irradiation activates endothelial cells which
then participate in the recruitment of circulating cells,
especially by overexpressing cell adhesion molecules but
also by other yet unknown mechanisms. In this study, we
aimed to determine whether irradiation modifies the
endothelium glycosylation pattern and to investigate the
impact of these changes on the adhesion of circulating
cells.
Material and Methods
Primary Human Umbilical Vein Endothelial Cells (HUVECs)
were irradiated at 20 Gy (
137
Cs source) and studied from