ESTRO 36 Abstract Book

S524 ESTRO 36 2017 _______________________________________________________________________________________________

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.

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 1 , 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

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 1 , M. Ben Kacem 1 , M. Dos Dantos 2 , F. Soysouvanh 1 , M. Benadjaoud 2 , A. Francois 1 , O. Guipaud 1 , F. Milliat 1 1 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 2 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

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