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Personalized Assessment of Normal Tissue Radiosensitivity via Transcriptome Response to Photon, Proton and Carbon Irradiation in Patient-Derived Human Intestinal Organoids

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Personalized Assessment of Normal Tissue Radiosensitivity via Transcriptome Response to Photon, Proton and Carbon Irradiation in Patient-Derived Human Intestinal Organoids

Ali Nowrouzi et al. Cancers (Basel).

Abstract

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.

Keywords: carbon ions; gastrointestinal (GI) toxicity; human intestinal organoids (HIO); particle therapy; personalized medicine; radiotherapy.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Irradiation of pluripotent derived human intestinal organoids with photon, proton and carbon irradiation. (a) Schematic outline on how organoids can be used to identify molecular surrogates of normal tissue radiation sensitivity and toxicity (b) Experimental outline of the study. Images of different stages of organoid development were kindly provided by the Center for Stem Cell and Organoid Medicine, Cincinnati. Human intestinal organoids (HIO) were generated as described previously and then embedded singularly in Matrigel in 96-plates. Following exposure to 4 Gy photon, proton and carbon irradiation HIO were cryo frozen for IF staining and stored for subsequent RNA isolation. (c) LEM prediction of carbon ions and positioning of organoids for irradiation. (d) Radiation-induced repair foci were visualized in cryo sections of irradiated HIO with IF stainings of 53BP1. The intensity of radiation-induced damage in HIO was quantified by measuring the 53BP1 numbers and size per cell. Represented values are normalized to background levels of 53BP1-foci.
Figure 2
Figure 2
Transcriptional patterns in response to photon, proton and carbon irradiation in wild type and FANCA-deficient HIO derived from healthy and Fanconi anemia individuals. The transcriptional status of genes related to the Fanconi anemia pathway was analyzed in the HIO-106 line 12 (a) and 48 h (b) post exposure to photon, proton and carbon ions. The Pearson correlation between wt and FANCA rescued and FANCA-deficient cells derived from FA patients clearly distinguishes FANCA-deficient HIO from HIO which express intact FANCA. For each time point gene expression values between healthy HIO and FA-derived 106-HIO (FANCA-deficient and FANCA rescued) were identified (p < 0.01, ANOVA; n = 1071 12 h; n = 1100 48 h) which are differentially regulated. All radiation qualities were combined for this analysis.
Figure 3
Figure 3
Differential expression of DNA damage response-related genes between wild type and Fanconi anemia (FA)-deficient HIO. (a) Represented are all the significantly enriched gene categories affected by genes which were significantly differentially regulated between wt and FA-deficient HIO both after 12 and 48 h. (b) The functional categories of genes which were time point specifically (12 and 48 h post IR) differentially regulated in FA-deficient cells were further divided. (c) Represented are the top differentially upregulated and downregulated genes for the 12 and 48 h time point separately and the gene list of genes commonly up- or downregulated is presented in the Venn diagram.
Figure 4
Figure 4
Gene expression changes as a function of Fanconi anemia, radiation quality and RBE. (a) gene expression heatmaps of genes which are correlated or anti-correlated with the higher RBE of carbon ions for the 12 and 48 h timepoint (p < 0.05, PTM). (b) The Venn diagram represent the overlap of genes modulated by FA, radiation qualities (RQ), and relative biological effectiveness (RBE) status for each timepoint resembling the core set of genes modulating FA response to radiation. (c) Genes which were modulated as a function of FA, RQ and RBE were analyzed with the STRING Search Tool for the Retrieval of Interacting Genes/Proteins. Represented are genes which interact with each other over the TP53 access.

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