ESTRO 36 Abstract Book

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S529 ESTRO 36
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involved in senescence process were measured by RT-
qPCR.
Results
While the clonogenic assay showed very similar survival
fraction curves for both conditions, we found highly
significant differences between the two conditions of
irradiation, when considering other biological outputs
when cell were irradiated at confluence. Cell number and
survival, morphological changes, cell cycle analysis,
molecular footprinting and β-galactosidase activity were
measured for doses up to 20 Gy. For all the assays, we
observed and demonstrated stronger effects on HUVECs
irradiated with the LINAC (4 MV) compared to the same
irradiation performed with the SARRP (220 kV).
Conclusion
All together these results strongly support the fact that
the clonogenic assay is not sufficient alone and that we
need to implement new models with multi-parametric
biological outputs to estimate a RBE that accurately
predicts the biological cellular fate. Such approach could
be useful for radiation protection but also for conditions
such as stereotactic body radiation therapy where the LQ-
model is inappropriate.
PO-0957 Radiobiological studies in in vitro
reconstituted squamous epithelia
G. Zemora1, W. Dörr 1
1 Medical University of Vienna, Department of
Radiotherapy- ATRAB - Applied and Translational
Radiobiology, Vienna, Austria
Purpose or Objective
Preclinical in vivo models are indispensable for
radiobiological investigations. However, their application
needs to follow the basic guidelines of animal studies
(reduction, refinement, replacement) and such research
should thus be supplemented by exploitation of suitable
alternatives, e.g. in vitro model systems. Three-
dimensional (3D) organotypic culture systems have been
shown to more accurately reflect the in vivo cell situation
as compared to standard 2D monolayer cell cultures. The
present study was initiated to generate and characterize
in vitro reconstituted human normal and malignant
squamous epithelia. These will then be applied for
analyses of the response to photon and ion irradiation, as
a basis for the design of subsequent in vivo studies.
Relevant damage processing pathways (including their
dependence on radiation quality) will be identified, and
biological targeting strategies will be screened. Also,
dedicated RBE studies at different positions in the ion
beam track for various endpoints will be performed.
Material and Methods
The 3D organotypic squamous epithelial tissues consist of
epithelial cells cultured on top of “dermal matrices”, i.e.
collagen gels formed from a collagen I solution populated
by metabolically active fibroblasts. Epithelial cells are
then seeded on top and cultured submerged. After 4 days
of submerged culture the gels are lifted so that the
epithelial cell monolayer is placed at the air-medium
interface and further cultured for 10 days until
stratification is complete. To reproduce skin equivalents,
HaCaT cells were seeded onto human skin fibroblasts gels
as mentioned above. To reconstruct normal and malignant
oral epithelia we will use immortalized normal oral and
FaDu squamous carcinoma cells, respectively. In
radiobiological studies, endpoints to be compared to the
in vivo situation will include morphology, differentiation,
DNA damage/repair (e.g. yH2AX, micronuclei) and various
radiation response-related signaling pathways, e.g of
inflammation through IL-6, TGF-ß and pro-MMP1. Single
dose as well as, importantly, daily fractionated irradiation
protocols will be applied for both photons and ions.
Results
Our preliminary efforts to reconstruct squamous epithelia
using HaCaT cells indicate the formation of a stratified
epithelium on top of a fibroblast-populated matrix. Our
results show positive IHC staining for the proliferating cells
expressing Ki67 located at the stratum basale, as well as
for the late differentiation proteins (involucrin and
loricrin). The experiments for the reconstruction of 3D
oral mucosa are ongoing.
Conclusion
In vitro reconstituted squamous epithelia are suitability
models, with intermediate complexity between 2D cell
cultures and tissues in vivo, for in vitro radiobiological
studies. Prospectively, macrophages will be integrated in
the 3D reconstructs to study radiation effects on the
immune system.
PO-0958 Radiogenomics: role of non-coding RNA genes
in increased radiotherapy sensitivity
L. Duran-Lozano1, V. Reyes2, M. Mollà2, M.J. Fuentes-
Raspall3, M. Altabas2, T. Ramón y Cajal 4, A. Barnadas4, O.
Diez1,5, J. Giralt2, S. Gutiérrez-Enríquez1
1Vall d'Hebron Institute of Oncology-VHIO, Oncogenetics
Group, Barcelona, Spain
2Vall d'Hebron University Hospital, Department of
Radiation Oncology, Barcelona, Spain
3Hospital de la Santa Creu i Sant Pau, Department of
Radiation Oncology, Barcelona, Spain
4Hospital de la Santa Creu i Sant Pau, Medical Oncology
Department, Barcelona, Spain
5Vall d'Hebron University Hospital, Area of Clinical and
Molecular Genetics, Barcelona, Spain
Purpose or Objective
Breast cancer (BC) is the first cause of cancer-related
mortality of Spanish women and most common cancer in
women worldwide. It is frequently treated with
radiotherapy (RT), which can cause early and late side-
effects that impact negatively on quality-of-life of cancer
survivors. MicroRNAs and long non-coding RNAs (lncRNAs)
modulate key cellular pathways in response to radiation.
Single nucleotide polymorphisms (SNPs) in these two types
of non-coding RNAs can alter their function and
consequently modify the expression of genes that
regulate, affecting the respective biological activities. As
part of a long-term ongoing study, our aims were to test
genetic association of SNPs in microRNAs and lncRNAs with
late radiotherapy-induced toxicity and to characterize the
expression of lncRNAs in blood cells of BC treated
patients. Our final goal is to discover new genetic
endpoints to predict individuals with increased
susceptibility to radiotherapy side effects.
Material and Methods
DNA samples and clinical data were collected from 198
prospectively and 72 retrospectively recruited BC patients
treated with RT in two hospitals. All patients were
followed at least between two and six years after RT. 34
SNPs in microRNAs and lncRNA genes related to radiation
response were genotyped using iPLEX® Gold with
MassArray Agena Bioscience (Sequenom). RNA was
obtained from blood before and after radiotherapy of 19
BC patients from the prospective cohort. Eight lncRNAs
(FAS-AS1, MALAT1, TP53TG1, HOTAIR, PANDA, MEG3,
ANRIL and LINC00467) involved in radiation cell response
were assessed by RT-PCR, agarose gels and direct
sequencing. A semiquantitative capillary electrophoresis
of fluorescent amplicons was performed to estimate the
proportion of total transcripts.
Results
The first analysis showed an association of overall long
term toxicity after radiotherapy with rs4559081 A/A
genotype of LINC00336 (OR=3.91 95%CI = 1.34-11.37), and
grade ≥ 2 late radiation skin toxicity (fibrosis or
telangiectasias) with rs17762938 A/C or CC of PCAT1
(OR=2.63 95% CI=1.00-6.86) and rs2910164 C/G of miR-
146a (OR=0.27 95%CI = 0.008-0.94). The expression and
presence of different isoforms of all lncRNAs evaluated,
except ANRIL and HOTAIR, were observed in blood cells.