Radiobiological Effects of Photon, Proton and Carbon Ion Irradiation on Human Pluripotent-Stem-Cell-Derived Gastrointestinal Organoids

Gastro-Intestinal (GI) toxicity constitutes an important obstacle in delivery of curative doses of radiotherapy. Advanced models recapitulating human GI physiology are urgently needed for a comprehensive radiobiological characterization of this radiosensitive organ system. Towards this goal, generation of organ like structures termed “organoids” from pluripotent stem cells is an emerging technology for dissecting normal tissue response and toxicity in a tissue specific context. Human gastrointestinal organoids (HIO) can be derived from patient specific or genome edited pluripotent stem cells. We report here a systematic characterization of HIO for photon- and particle therapies (proton and carbon ion irradiation). Morphological, functional and molecular effects of the three radiation qualities was investigated by means of immunohistology, DNA damage repair kinetic and genome-wide transcriptional response (mRNA and microRNA). Irradiation induced DNA-double-strand-breaks were successfully detected in cryopreserved organoids by immunohistochemical staining of 53BP1 foci enabling the analyses of DNA repair kinetics in different mature intestinal cell types present in HIO. Following unsupervised hierarchical clustering of gene expression signatures we selected for the strongest differences within the irradiated compared to non-irradiated HIO. In order to identify early- and late transcriptional changes in response to photon and particle therapy z-transformed expression values were compared 12 and 48 hours following irradiation. This comparison revealed that the strongest differences in gene expression detected in organoids are enriched in particular radiotherapy relevant pathways and can distinguish radiation qualities as well as dynamics of early and late transcriptional changes following irradiation. Human pluripotent-stem-cell-derived organoids therefore provide an attractive model to study radiotherapy response and toxicity independent of animal models.