Characterization of irradiated colonic stroma for therapeutic treatment of Pelvic Radiation Disease
OC-0599
Abstract
Characterization of irradiated colonic stroma for therapeutic treatment of Pelvic Radiation Disease
Authors: Martin Jestin1, Noëlle Mathieu1, Claire Squiban1, Christelle Demarquay2, Fabien Milliat3, Mohamedamine Benadjaoud4
1Institute for Radiological Protection and Nuclear Safety, Radiobiology of Medical Exposure Laboratory, Fontenay-aux-Roses, France; 2Institute for Radiological Protection and Nuclear Safety, Radiobiology of Medical Exposure Laboratory , Fontenay-aux-Roses, France; 3Institute for Radiological and Nuclear Safety , Radiobiology of Medical Exposure Laboratory, Fontenay-aux-Roses, France; 4Institute for Radiological and Nuclear Safety, Radiobiology and Regenerative Medicine Research Service, Fontenay-aux-Roses, France
Show Affiliations
Hide Affiliations
Purpose or Objective
Pelvic cancers have a high prevalence and are mainly treated by radiotherapy. While it provides tumor control, it also causes damage to surrounding healthy tissues, leading to disabling complications defined as the Pelvic Radiation Disease (PRD). Today, no curative treatment exists for this fibrosing pathology. This project aims to study the colonic microenvironment (µE) after irradiation and identify new target to improve the management of PRD in the colon.
Material and Methods
We use a mouse model developing fibrotic colonic damage like those observed in patients with PRD. This model consists of a single colorectal irradiation of 26Gy. Two study times were defined after irradiation: at two weeks (W2) to study acute effects of irradiation; at twelve weeks (W12) to explore fibrosis. Histological immunostaining was performed to assess proliferation (Ki67) and differentiation (MUC2; ChgA) capacities of the colonic epithelium as well as expression of epithelial junction proteins (ZO-1; P120/cat). We then studied in vitro the impact of the colonic µE on epithelial proliferation. To this aim, colonic organoids were cultured in presence of sorted CD45- & EpCAM- stromal cells from irradiated or unirradiated mice at W2 & W12. Colonoids development was assessed according to , number and type of colonoids. Finally, colonic µE remodeling was studied at the molecular level on sorted stromal cells by single-cell transcriptomic analysis at W2 & W12.
Results
Based on immunohistochemistry, we observed epithelial junctions impaired after irradiation at W2 & W12. However, epithelial proliferation and differentiation are increased at W2 after irradiation at the lesion’s edge. Interestingly, this rise persists and even increased at W12 leading to anarchic crypt structures. Coculture experiments show a higher growth of colonoids when cocultured with stromal cells. Among coculture conditions, stromal cells from W12-irradiated mice allow the greatest growth of colonoids in term of and number. Single-cell transcriptomic analysis allowed the identification of different populations constituting the colonic µE based on the literature. We also highlighted an undefined fibroblastic population. Moreover, analyzes carried out at the level of specific populations have defined molecular modifications after irradiation.
Conclusion
After irradiation, colonic epithelium shows impaired barrier, however a regenerative process with enhanced proliferation and differentiation is set up at W2 and still rise at W12. Coculture results demonstrate that stroma promotes colonoids development with a significant impact of stromal cells from irradiated mice W12. These results suggest the direct role of W12-irradiated stromal cells on epithelial disorders. Analyzes carried out at the transcriptomic level have defined cellular and molecular pathways modifications that could be involved in epithelial dysfunctions and explained coculture results. These pathways could be modulated to reduce colonic damage after irradiation.