ESTRO 37 Abstract book

S303

ESTRO 37

Purpose or Objective Radiotherapy is indicated for the treatment of half of all cancer patients. However, incidental irradiation (IR) of the bowel results in dose-limiting side effects in the majority of patients receiving abdominal or pelvic radiation treatment. The major late stage complication in the irradiated bowel is connective tissue fibrosis. In the intestine, this process is likely driven by the immune response in the acute phase through the activation and release of both pro- and anti-inflammatory cytokines, such as IL-6, TNF-a, IL-1b, IL-10, following focal IR. The purpose of this study is to determine the role of the immune system in the promotion of intestinal fibrosis following focal IR and to propose potential targets for blocking the development of fibrosis. Material and Methods We recently developed a mouse model of image-guided, radiation-induced intestinal damage which can serve as a new tool for precise and quantitative evaluation of multiple biological markers. It involves surgically implanting a radiopaque marker onto the surface of the jejunum in order to replicate highly focal clinical radiotherapy. Mice were then imaged with cone beam computed tomography to locate the marker, and irradiated with 18 Gy of 5x5 mm collimated X-rays onto the marked intestine using the Small Animal Radiation Research Platform (SARRP). Results Irradiated mice exhibited 100% survival with minimal weight loss after IR when compared to mock-irradiated controls. Irradiation significantly impaired crypt regeneration as assessed by a 5-ethynyl-2’-deoxyuridine (EdU) cell proliferation assay at day 1 post-IR. Furthermore, a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay indicated a localized increase in crypt apoptosis in the IR area, suggesting the marked intestine was successfully irradiated. Beginning 3.5 days post-IR, there was significant ablation of intestinal structure with very few proliferating cells in the IR area. The intestinal barrier was maintained in the acute phase by markedly hyperplastic crypts on the edges of the IR area. Furthermore, a severe innate immune response was observed in the irradiated intestine 3.5, 7, and 14-days post-IR as evidenced by increased neutrophil and macrophage infiltration. This inflammatory process induced a systemic leukocytosis which was driven by an increase in neutrophils in the peripheral blood. Using an ELISA, to determine the levels of inflammatory cytokines post-IR, we observed a significant increase in Interleukin- 6 (IL-6) both in the irradiated intestine and in the blood plasma (3.5d post-IR). Furthermore, we identified using IL-6 -/- mice that IL-6 was critical for survival post 18 Gy of focal irradiation as evidenced by 70% of IL-6 deficient mice dying due to intestinal perforation. Two-months post-IR, fibrosis was observed in the irradiated area by Masson’s trichrome stain. Conclusion Our results suggest that a major inflammatory reaction in the acute phase precedes fibrosis, with the IL-6 be the critical molecule in this process. and ceramide contribute to radiation-induced intestinal toxicity H. Estéphan 1 , D. Leonetti 1 , M.H. Gaugler 1 , E. Hadchity 2 , I. Corre 1 , F. Paris 1 1 INSERM- UMR 892, Equipe 14 Radiobiologie et ciblage de l'endothélium, Nantes, France 2 Anti-Tumor Therapeutic Targeting Laboratory- Faculty of Sciences, Lebanese University, Hadath, Lebanon Purpose or Objective A single high dose of 15 Gy induces endothelial cell (EC) apoptosis through the activation of acid sphingomyelinase OC-0585 Secreted acid sphingomyelinase

(ASM) and the generation of the proapoptotic sphingolipid ceramide leading to intestinal collapse. The mode of action of EC apoptosis in the intestinal epithelial radiosensitivity is not defined.In the present study, we evaluated whether systemic ASM and ceramide acutely secreted by irradiated endothelial cells, represent paracrine factors enhancing irradiation (IR)-induced toxicity in the small intestine. Material and Methods ASM and ceramide secretion were assessed respectively by enzymatic assay and LC-MS/MS in serum from wild type (WT) and ASMKO C57BL/6 mice, then in supernatants of primary human lung microvascular EC (HMVEC-L), after 15 Gy. Involvement of ASM and ceramide in human intestinal epithelial T84 cell growth and apoptosis were quantified after exogenous treatment by cell counting and Cell Death Detection ELISA kit respectively. To definitely validate the ASM/ceramide secreted by EC in intestines epithelial cell radiosensitivity, a co-culture model without contact using Transwell® dish has been assessed. T84 cells and EC transfected with either sham or ASM siRNA were irradiated at 15 Gy together or separately, cell growth and apoptosis were studied. Results Exposure to 15 Gy in WT mice induced small intestines collapse and a significant increase of secreted ASM and ceramide in serum respectively at 30 min and 24h up to 96h post-IR. On the other hand, seric ASM and ceramide were not increased in 15 Gy-IR ASM-KO mice, in correlation with limited intestinal injury. Then, a cell culture study was developed to validate the involvement of secreted ASM/ceramide in the intestinal epithelial cell radiosensitivity. First, ASM and ceramide secretion were observed in EC, 24 and 72h after 15 Gy. Then, exogenous ASM or ceramide treatment were able to resume cell growth arrest and death observed when intestinal epithelial T84 are in co-culture with irradiated EC. Finally, invalidation of ASM in irradiated EC abolished the bystander response on the T84 cells. Conclusion Our study highlights the role of ASM and ceramide as paracrine factors in radiation-induced intestinal toxicity. Endothelial cells are a main source of their secretion suggesting the need to protect these cells from the toxic effects of radiation in order to limit intestinal failure. OC-0586 Improving lung cancer outcome by reducing normal lung toxicity L. Giuranno 1 , E. Moreno Roig 1 , R. Iannone 1 , C. Wansleeben 1 , M. Vooijs 1 1 Maastricht Radiation Oncology MAASTRO, Radiotherapy, Maastricht, The Netherlands Purpose or Objective Lung cancer is the leading cause of cancer death in western countries. The current standard of care includes surgery , chemotherapy and radiotherapy. While significant progress has been made in terms of treatment modality radiotherapy is limited by dose-limiting side- effects which negatively affect tumour control and patient's quality of life. Reducing side-effects may improve tumor control by dose-escalation and treatment- time. The Notch signaling pathway plays an important role in lung cell differentiation and regeneration of the airway epithelium and its deregulation is associated with several cancers including lung cancer (Lyo, 2016). Notch signaling pathway alteration leads to poor outcome and treatment resistance in patients and in preclinical models (Theys, 2013) suggesting Notch signaling as a novel therapeutic target. However, the mechanism through which Notch inhibition integrates with airway repair and cellular differentiation is not fully understood (Tsao, 2011).What is currently lacking are primary human lung tissue models that enable robust evaluation of normal