Ali Nowrouzi, Mathieu G Sertorio, Mahdi Akbarpour, Maximillian Knoll, Damir Krunic, Matthew Kuhar, Christian Schwager, Stephan Brons, Jürgen Debus, Susanne I Wells, James M Wells, Amir Abdollahi

Radiation-induced normal tissue toxicity often limits the curative treatment of cancer. Moreover, normal tissue relative biological effectiveness data for high-linear energy transfer particles are urgently needed. We propose a strategy based on transcriptome analysis of patient-derived human intestinal organoids (HIO) to determine molecular surrogates for radioresponse of gastrointestinal (GI) organs at risk in a personalized manner. HIO were generated from induced pluripotent stem cells (iPSC), which were derived from skin biopsies of three patients, including two patients with FANCA deficiency as a paradigm for enhanced radiosensitivity. For the two Fanconi anemia (FA) patients (HIO-104 and 106, previously published as FA-A#1 IND-iPS1 and FA-A#2 IND-iPS3), FANCA expression was reconstituted as a prerequisite for generation of HIO via lentiviral expression of a doxycycline inducible construct. For radiosensitivity analysis, FANCA deficient and FANCA rescued as well as wtHIO were sham treated or irradiated with 4Gy photon, proton or carbon ions at HIT, respectively. Immunofluorescence staining of HIO for 53BP1-foci was performed 1 h post IR and gene expression analyses was performed 12 and 48 h post IR. 53BP1-foci numbers and size correlated with the higher RBE of carbon ions. A FANCA dependent differential gene expression in response to radiation was found (p < 0.01, ANOVA; n = 1071 12 h; n = 1100 48 h). Pathways associated with FA and DNA-damage repair i.e., transcriptional coupled nucleotide excision repair, homology-directed repair and translational synthesis were found to be differentially regulated in FANCA deficient HIO. Next, differential regulated genes were investigated as a function of radiation quality (RQ, p < 0.05, ANOVA; n = 742 12 h; n = 553 48 h). Interestingly, a gradual increase or decrease of gene expression was found to correlate with the three main qualities, from photon to proton and carbon irradiation. Clustering separated high-linear energy transfer irradiation with carbons from proton and photon irradiation. Genes associated with dual incision steps of TC-NER were differentially regulated in photon vs. proton and carbon irradiation. Consequently, SUMO3, ALC1, POLE4, PCBP4, MUTYH expression correlated with the higher RBE of carbon ions. An interaction between the two studied parameters FA and RQ was identified (p < 0.01, 2-way ANOVA n = 476). A comparison of genes regulated as a function of FA, RQ and RBE suggest a role for p53 interacting genes BRD7, EWSR1, FBXO11, FBXW8, HMGB1, MAGED2, PCBP4, and RPS27 as modulators of FA in response to radiation. This proof of concept study demonstrates that patient tailored evaluation of GI response to radiation is feasible via generation of HIO and comparative transcriptome profiling. This methodology can now be further explored for a personalized assessment of GI radiosensitivity and RBE estimation.