DOP110 Optimized inflammatory and ER stress-induced phenotypes in differentiated intestinal organoids for modelling Inflammatory Bowel Disease

Abstract Background Differentiated intestinal organoids (ORG), which retain patient- and disease-specific features, offer a powerful model to study inflammatory bowel disease (IBD) mechanisms. While ORG preserve IBD characteristics, acute inflammation fades in culture. To address this, we developed a series of inflammatory mixes to reintroduce disease phenotypes. Given that IBD pathways also involve endoplasmic reticulum (ER) stress—a factor in pathogenesis and a potential therapeutic target—this study aimed to optimize two specific mixes: one to induce a purely inflammatory (INF) response and another to simulate ER stress-driven inflammation in differentiated intestinal organoids. Methods Biobanked intestinal ORG cultures, derived from non-inflamed endoscopy biopsies from UC patients (n=9), were expanded, mechanically split, and exposed to human expansion media (HM) for two days1. Next, ORG cultures were exposed to differentiation media (DM) for four days. From day 6 onwards, ORG were maintained in DM only, or supplemented with an INF mix (50 ng/ml TNF-α, 20 ng/ml IL-1β, 1 µg/ml Flagellin) or an ER stress mix (10 ng/ml TNFα, 20 ng/ml IL-1β, 1 µg/ml Flagellin, Tunicamycin (TM) 25ng/ml) for 48 hours (Figure1A). As TM can induce both inflammatory and ER stress pathways, a concentration range of TM (25 ng/ml – 1 µg/ml) and TNF-α (10 ng/ml – 100 ng/ml) has been evaluated by cell morphology to determine the threshold without comprising cell viability (Figure1B). Results Principal component analysis (PCA) demonstrated distinct clustering by treatment condition, with clear separation observed across all groups (Figure2A-B).PCA plots showed consistency with the directionality of key genes driving the PC. For instance, the upregulation of PLAU2 in the ER and INF-mix groups contributes to the separation along PC1, PSAT13, drives the separation along PC2, aligning with the ER mix group, and CCL204 is instead driving PC3 (Figure2C). In organoids treated with the ER stress mix, multiple ER stress markers showed strong upregulation compared to those exposed to the INF mix, with a more pronounced effect in terms of significance and fold change (Figure2D). Pathway analysis of the contrast of the NO-mix versus the ER mix further confirmed the activation of ER stress-related pathways (Figure2E). Conclusion We developed two mixes to model INF and ER stress in differentiated intestinal ORG. PCA analysis confirmed distinct clustering by condition, with key genes driving group separations. In addition, a stronger upregulation of ER stress markers and pathways was observed in the ORG treated with the ER-mix. This newly designed mixes will facilitate preclinical compound evaluation in a disease representative IBD model. References 1.Arnauts et al., Gastroenterology, 2020 ; 2.Cheng, Yang et al. eBioMedicine, 2022 ; 3. Reich S, et al Nat Commun,2020 ; 4.Skovdahl HK, et al. PLoS One. 2015 Giorio and Arnauts are shared first author.