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3 Université Catholique de Louvain, Radiobiology Laboratory, B-1200 Brussels, Belgium 4 iThemba LABS, Department of Radiation Biophysics, 7129 Somerset West, South Africa Purpose or Objective The stem cell (SC) population in the intestinal crypts (IC) is a field of intense research, especially with regard to the role of SC's in tissue repair. It is even more relevant in radiation-oncology as the intestine presents an organ at risk. We have recently observed a strong volume effect in the survival of intestinal ICs after irradiation of different lengths of externalized mouse jejunum. Here, we present a biomathematical model that reproduces this volume effect to great precision by an increase in proliferation rate for small irradiated volumes and which, applied in a meta-analysis, resolves current issues on number and radio-sensitivity of reserve SCs. Material and Methods Interpretation of the in vivo clonogenic assay is commonly obtained via a formula that relates IC survival to the number of clonogenic cells per IC and their radio- sensitivity. This IC survival model is the only one used up to date despite questionable underlying assumptions and highly inconsistent results between studies. Our model calculates the full statistics of IC procreation over time on a cell level, which allows a strict model validation on several additional experimental levels beyond the mere IC count, such as cell-population-per-IC statistics and spatial distribution of surviving ICs. Results We could successfully test the model on a large dataset: IC survival for three different irradiated volumes (whole- bowel, 10mm and 5mm jejunum) at 4-8 irradiation doses; spatial distribution of IC survival at different doses; distribution of cell survival per IC at different doses and volumes. We observed no sign for a migration mechanism of SCs in our data, as all datasets could be reproduced by statistical variation. This might hint towards a purely stochastic repopulation mechanism in the first days of regeneration. The model reproduced precisely the volume effect on all of these levels if an increase in the PR was assumed while all other parameters remained unchanged (Fig. 1). The volume effect is observed for the 5mm, but not for the 10mm field, which indicates a threshold volume, in accordance with volume effects in other organs.

Conclusion Our model dramatically increases the precision of the clonogenic assay and turns it into a precision tool for the investigation of SC procreation. It explains the small-field volume effect in intestinal epithelium as accelerated repopulation. Our experiments show that below a threshold volume, the overall proliferation rate drastically increases. The results from the meta-analysis resolve a persisting conflict in the predicted size of the reserve SC population and the radio-sensitivity of reserve SCs. This creates new opportunities for high-precision analysis of e.g. the effects of biochemical agents. OC-0584 Acute changes in leukocyte populations driving fibrosis following focal irradiation of the intestine I. Verginadis 1 , B. Bell 1 , S. Koduri 1 , C. Koumenis 1 1 Perelman School of Medicine- University of Pennsylvania, Department of Radiation Oncology, Philadelphia, USA 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 wi th 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

We applied this model in a meta-analysis of a large number of data from the last four decades. The results now give a consistent picture of a precise number of clonogenic cells of 31±6, which are, in contrast to prior predictions, radio-resistant (α = (0.21±0.02)Gy^-1) (Fig. 2).