Abstract
5-Fluorouracil (5-FU) is a widely used chemotherapeutical that induces acute toxicity in the small and large intestine of patients. Symptoms can be severe and lead to the interruption of cancer treatments. However, there is limited understanding of the molecular mechanisms underlying 5-FU-induced intestinal toxicity. In this study, well-established 3D organoid models of human colon and small intestine (SI) were used to characterize 5-FU transcriptomic and metabolomic responses. Clinically relevant 5-FU concentrations for in vitro testing in organoids were established using physiologically based pharmacokinetic simulation of dosing regimens recommended for cancer patients, resulting in exposures to 10, 100 and 1000 µM. After treatment, different measurements were performed: cell viability and apoptosis; image analysis of cell morphological changes; RNA sequencing; and metabolome analysis of supernatant from organoids cultures. Based on analysis of the differentially expressed genes, the most prominent molecular pathways affected by 5-FU included cell cycle, p53 signalling, mitochondrial ATP synthesis and apoptosis. Short time-series expression miner demonstrated tissue-specific mechanisms affected by 5-FU, namely biosynthesis and transport of small molecules, and mRNA translation for colon; cell signalling mediated by Rho GTPases and fork-head box transcription factors for SI. Metabolomic analysis showed that in addition to the effects on TCA cycle and oxidative stress in both organoids, tissue-specific metabolic alterations were also induced by 5-FU. Multi-omics integration identified transcription factor E2F1, a regulator of cell cycle and apoptosis, as the best key node across all samples. These results provide new insights into 5-FU toxicity mechanisms and underline the relevance of human organoid models in the safety assessment in drug development.
Highlights
5-Fluorouracil (5-FU) is a chemotherapeutic agent that belongs to the class of fluoropyrimidines, the fluorinated derivatives of pyrimidines, and to the group of antimetabolite drugs
Organoids derived from biopsies of the small intestine (SI) and colon of healthy human donors were successfully generated in a 3D culture matrix
Both types of organoids were exposed to concentrations of 5-FU derived from physiologically based pharmacokinetic (PBPK) modelling to mimic the in vivo concentrations following clinically relevant dosing
Summary
5-Fluorouracil (5-FU) is a chemotherapeutic agent that belongs to the class of fluoropyrimidines, the fluorinated derivatives of pyrimidines, and to the group of antimetabolite drugs. Intestinal mucositis is usually accompanied by nausea, abdominal pain, vomiting and diarrhoea, which has a major impact on health and the quality of life of patients These ADRs and other complications, namely dose-limiting diarrhoea, may lead to the interruption or alteration of the cancer therapy (Dore et al 2017). The use of intestinal organoids has increased exponentially in recent years and has shown potential for the investigation of intestinal development, homeostasis, and human disease development These organoids have not yet been fully explored for the evaluation and screening of drug toxicity. This study aimed at evaluating whether human intestinal organoids are a suitable model to investigate the mechanism of action underlying 5-FU induced intestinal toxicity by identifying differentially expressed genes (DEGs) and the most affected biological pathways, which can be translated to functional endpoints and human responses
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