ESTRO 36 Abstract Book

S524

ESTRO 36 2017

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Results of this study constitute a first approach to

demonstrate the therapeutic benefit of MSC infusion on

vascular compartment in a model of severe colonic

damages induced by radiations. We also characterized the

molecular mechanisms involved in regenerative capacities

of MSC and determined that the limitation of the vascular

permeability could be a way of therapeutic improvement.

This cell and pharmacologic co-treatment could be used

for compassionate applications to reduce colorectal

damages induced by pelvic radiotherapy.

Poster: Radiobiology track: Normal tissue radiobiology

(others)

PO-0956 Prediction of irradiated cells fate: the

necessity to revisit RBE by multi-parametric

investigations

V. Paget

, M. Ben Kacem

, M. Dos Dantos

, F.

Soysouvanh

, M. Benadjaoud

, A. Francois

, O. Guipaud

F. Milliat

Institute for Radiological Protection and Nuclear Safety

IRSN, Department of Radiobiology and Epidemiology

SRBE / Research on Radiobiology and Radiopathology

Laboratory L3R, Fontenay-aux-Roses- Paris, France

Institute for Radiological Protection and Nuclear Safety

IRSN, Department of Radiobiology and Epidemiology

SRBE, Fontenay-aux-Roses- Paris, France

Purpose or Objective

The evaluation of radiosensitivity is historically linked to

the survival fraction measured by the clonogenic assay,

which is until now the gold standard in such evaluation.

The representation of the survival fraction as a function

of the dose leads to survival curves which are modelled by

the linear-quadratic model (LQ-model). The Relative

Biological Effectiveness (RBE) is defined as the ratio of the

doses required by two types of ionizing radiations to cause

the same biological effect (for instance the survival

fraction). The RBE is an empirical value that varies

depending on the type of particle, the Linear Energy

Transfer (LET), the dose rate and the dose fractionation,

and can be easily used to predict biological outcome in

different situations. Nevertheless, the clonogenic assay is

a quite restrictive method which does not take into

account cell-cell interactions and the phenotype of

surviving cells as well. Thus, the aim of this study is to

demonstrate, by a proof of concept, the limits of the

clonogenic assay to predict the cellular fate and in a lesser

extend its unsuitability to predict accurately on healthy

tissues the risk associated to the use of ionizing radiations.

Material and Methods

Radiation-induced damage to the vascular endothelium is

potentially involved in the initiation and the development

of normal tissue injury. Thus, in this study we compared

the biological effects on HUVECs (Human Umbilical Vein

Endothelial Cells) exposed to low energy x-rays (generated

at 220 kV on a SARRP) and high energy x-rays (generated

at 4 MV on a LINAC). Cell survival fractions were

measured/calculated by using clonogenic assay while

morphological changes, cell viability/mortality, cell cycle

analysis and β-galactosidase activity were evaluated by

flow cytometry. Finally molecular footprinting of 44 genes

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. Zemora

, W. Dörr

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