P263 Irinotecan induces a rapid increase in enteroid permeability

Introduction Chemotherapy-induced diarrhoea is a common side effect for patients given irinotecan and can result in the cessation/interruption of treatment affecting clinical efficacy. The clinical approach is to treat diarrhoea symptomatically as there are currently no preventative therapies. Recent in vivo studies suggest that irinotecan initiates two phases of diarrhoea with the first attributed to changes in intestinal permeability through tight junction (TJ) disruption. We have now investigated whether small intestinal (SI) organoids (enteroids) can be used to model SI permeability changes induced by irinotecan to dissect the mechanisms responsible for irinotecan-induced diarrhoea. Understanding these mechanisms may enable novel approaches to reduce intestinal toxicity. Methods Proximal SI derived enteroids from C57BL/6 wild-type mice were treated with 0–100 μM irinotecan and imaged daily for 96 hrs. Enteroid circularity (4π(area)/perimeter2) was measured as a marker of enteroid health and active caspase-3 IHC was used to assess apoptosis. Enteroids were microinjected with 1 mg/ml Texas Red and treated 30 mins post injection with 100µM irinotecan or 5 mM EGTA. Fluorescent images were taken hourly for 4 hrs. Mean pixel intensity was measured after injection. The minimum threshold was set by mean intensities of untreated, none injected enteroids. Subsequent time point mean pixel intensity was expressed as a percentage of immediate post injection intensity. Images were manually quantified to validate the method. Results Healthy enteroids maintained circularity values of 0.38±0.06. Irinotecan caused dose and time dependant increases in enteroid circularity with maximal rounding at 100 μM by 48 hrs (0.75±0.05). Dose and time-dependent increases in active caspase-3 were observed. Microinjection assays were optimised to assess very early effects of irinotecan on SI permeability. Control enteroids stabilised to 71.33±8.5% starting intensity at 1 hr, EGTA (positive control) dropped to 31.31±1.97% and 100 μM irinotecan reduced mean intensity to 46.04±3.71% after 30 mins. Area under the curve (AUC) for 0–4 hrs post-treatment showed statistically significant increased SI permeability for irinotecan (p Conclusion Irinotecan caused a rapid onset of SI barrier dysfunction in enteroids suggesting that this precedes irinotecan-induced apoptosis and may be in part due to the disruption of TJs. Further investigation is now needed to determine whether pre-treatment with TJ stabilising drugs may ameliorate irinotecan-induced permeability and diarrhoea.