Development of a self-limiting model of Methotrexate-induced mucositis reinforces butyrate as a potential therapy

Abstract

Background: Gastrointestinal mucositis remains a significant complication of anticancer treatment, with limited anti-mucositis interventions. Currently, there are few validated in vitro systems suitable to study the mechanisms of mucosal injury. Therefore, we aimed to develop and characterize a chemotherapeutic-induced model of mucositis using 3D intestinal organoids. Methods: Organoids derived from mouse ileum were grown for 7 days and incubated with different concentrations of the chemotherapeutic agent methotrexate (MTX), ranging from 0-1000 ng/ml. Metabolic activity, citrulline levels and cytokine/chemokine production were measured to determine the optimal dosage and incubation time. To link the model to clinical practices, the protective effects of folinic acid on the toxicity of MTX were investigated by pre-treating organoids with (0.0005-50 µg/mL) folinic acid. Given the impact of microbial-derived short-chain fatty acids in epithelial health, we also evaluated the impact of short-chain fatty acid supplementation on MTX toxicity in the organoid model. Results: MTX treatment caused a dose-dependent reduction in cell metabolic activity and citrulline production. Folinic acid treatment reduced MTX toxicity when applied simultaneously or up to 24 hours after treatment. Supplementation of the growth medium with butyrate reduced MTX toxicity. Analysis of organoid gene expression suggests that butyrate may reduce intracellular MTX concentrations by increasing the expression of MTX transporters, including the ABCC1 transporter.Conclusion: MTX causes significant organoid damage, which can be reversed upon removal of MTX. The specific readouts and the protective effects of folinic acid suggest that the model is clinically relevant, and can be used in the future to study mucositis, diminishing the need for animal models. By using the model for intervention, it was found that prophylactic treatment with butyrate might be a valuable strategy for prophylactic mucositis prevention.