Abstract 2125: The use of intestinal organoids as a preclinical screen to assess gastrointestinal (GI) toxicity and barrier integrity

Abstract

Abstract Gastrointestinal (GI) toxicity is a common and often severe limiting factor in the development of antineoplastic drugs. Symptoms include diarrhoea, dehydration and ulceration which increase susceptibility to infection, partly due to damage of crypt and/or villus structures in the small intestine, impairing the barrier function. As novel targeted therapies are emerging, assessment of their potential GI toxicity remains crucial. Small intestinal (SI) organoids are 3D in vitro models of the epithelium which recapitulate the structure and function of the small intestine. We have further developed and validated the organoid model as a screening tool to predict GI toxicity and subsequent mucosal regeneration in four species: mouse, rat, dog and human. The culture conditions have been designed to mimic the stem cell niche and allow cell differentiation and proliferation to occur. All intestinal lineages were present in the organoids derived from each species and the epithelial hierarchy closely resembled that observed in vivo. The toxic effects of antineoplastic drugs on the organoids were assessed by quantification of the total cell viability upon treatment, coupled to morphometric analysis of the organoids (size, area, perimeter, and branching efficiency). Immunofluorescence and RNAseq can be used to identify the targeted cells and the toxicity mechanisms of the drugs. To further assess the effect of drugs on barrier integrity, we have developed transwell-grown cell monolayers derived from normal human small intestinal organoids. This model allows direct access to both apical and basal surfaces, which is not possible when using organoids where their geometry prevents access to the apical surface. The monolayer formation was followed by live imaging and Trans-Epithelial Electrical Resistance (TEER) measurements and treatments with toxic drugs were applied after TEER reached a plateau. The direct effect on barrier integrity was evaluated by TEER reduction, increased FITC-Dextran permeability and analysis of TJ proteins by immunofluorescence. Furthermore, effects on cell death were assessed by measuring the levels of Lactate Dehydrogenase (LDH) released into the medium by dying cells. In conclusion, our models provide an innovative in vitro solution to further study the toxic effects of drugs on the normal small intestine and to analyse direct effects on barrier integrity. The monolayer assay may also be useful for developing models of transient TJ impairment to improve the bioavailability of orally administered antineoplastic drugs. The model can be considered more representative of the normal intestine than transformed cell lines such as CaCo2. Citation Format: Valentina Ubertini, Sarah Hall, Frida Ponthan, James Wilson. The use of intestinal organoids as a preclinical screen to assess gastrointestinal (GI) toxicity and barrier integrity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2125.