New insights into the mechanisms underlying 5-fluorouracil-induced intestinal toxicity based on transcriptomic and metabolomic responses in human intestinal organoids

Abstract

Abstract5-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.

Unravelling Mechanisms of Doxorubicin-Induced Toxicity in 3D Human Intestinal Organoids

“…Colon and SI organoids were also grown in Human IntestiCult™ Organoid Growth Medium (Stemcell, Cologne, Germany), prepared according to manufacturer's instructions, to promote organoid differentiation. Differentiation was verified after 2-3 days, showing that organoids started to form buds that resembled intestine-like features, as described in our previous study [10].…”

“…For this reason, this study aimed at not only confirming the hypothetical mode of action of the drug, but mainly at generating new data to advance our knowledge of the mechanisms involved in DOX-induced intestinal toxicity. To accomplish this, high-throughput transcriptomic analysis was performed on innovative 3D culture models of colon and small intestine (SI) organoids, derived from human tissue biopsies [10].…”

A Transcriptomic Approach to Elucidate the Mechanisms of Gefitinib-Induced Toxicity in Healthy Human Intestinal Organoids

“…In this study, we investigated the molecular underlying the toxic effects of gefitinib in a newly established 3D culture of healthy human intestinal organoids derived from colon and SI tissues. These in vitro models have been developed to better resemble the characteristics and responses of the human tissues [18,38,39] and they have been successfully applied in the research of drug toxicity induced by 5-FU [14] and doxorubicin [40]. PBPK modelling was used in order to determine in-vitro drug concentrations that are clinically relevant.…”

“…In this study, three-dimensional (3D) innovative cell models of human colon and small intestine (SI) were established as described in our previous work [14] due to their potential in drug-response modulation. The establishment of 3D organoid systems has exponentially increased in recent years as they replicate in vivo cellular organization, behaviour, and cellenvironment interactions more closely than conventional 2D cultures [15,16].…”

“…Responses in colon and SI tissues were compared to establish if their different cell composition, dynamics and function is reflected by distinct gene expression profiles. The exposure concentrations for the in vitro experiments with gefitinib were based on physiologically based pharmacokinetic (PBPK) modelling and simulation to guarantee that the in vitro concentrations of gefitinib reflect patients' dose regimens during chemotherapy [14,26]. Intestinal organoids were exposed to four different concentrations (0.1, 1, 10 and 30 µM) for three days.…”