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

Abstract

Gastrointestinal mucositis is a complication of anticancer treatment, with few validated in vitro systems suitable to study the complex mechanisms of mucosal injury. Therefore, we aimed to develop and characterize a chemotherapeutic-induced model of mucositis using 3D intestinal organoids. Organoids derived from mouse ileum were grown for 7 days and incubated with different concentrations of the chemotherapeutic agent methotrexate (MTX). Metabolic activity, citrulline levels and cytokine/chemokine production were measured to determine the optimal dosage and incubation time. 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. The impact of microbial-derived short-chain fatty acids was evaluated by supplementation with butyrate in the organoid model. MTX caused a dose-dependent reduction in cell metabolic activity and citrulline production that was salvaged by folinic acid treatment. Overall, MTX causes significant organoid damage, which can be reversed upon removal of MTX. The protective effect of folinic acid suggest that the organoids respond in a clinical relevant manner. By using the model for intervention, it was found that prophylactic treatment with butyrate might be a valuable strategy for prophylactic mucositis prevention.

Figure 1

Evaluation of methotrexate-induced cytotoxicity in organoids. Organoids were treated with different concentrations of MTX (0, 10, 100 and 1000 ng/mL) for different time points (24, 48, 72 and 96 h). (A) Relative metabolic activity of organoids treated with or without MTX (n = 6). (B) Citrulline levels in culture supernatants collected at 24, 48, 72 and 96 h of incubation with MTX (µmol/l) (n = 4). (C) Optical microscopy of MTX-treated organoids for 96 h (40x). Scale bar = 200 μm. All data are presented as mean ± SEM. *(p < 0.05), **(p < 0.01) and ***(p < 0.001) represent the significant changes between MTX-treated and untreated organoids.

Figure 2

Relative metabolic activity after media refreshment. After 96 h of incubation, medium containing MTX was washed away and fresh media was added. Supernatant was collected at 24, 48, 72 and 96 h and WST-1 performed (n = 3). All data are presented as mean ± SEM. * (p < 0.05), ** (p < 0.01) and *** (p < 0.001) represent the significant changes between MTX-treated and untreated groups.

Figure 3

Folic acid dose response in MTX-induced mucositis organoids. Relative metabolic activity of organoids treated with 100 ng/mL MTX and the simultaneous addition of 0.005, 0.05, 0.5, 5 and 50 µg/mL folinic acid for 96 h (n = 3). Data is presented as mean ± SEM. #p < 0.05 for control organoids versus MTX-treated organoids. *p < 0.05 and p < 0.0001 for MTX-treated organoids versus MTX and FA-treated organoids.

Figure 4

Relative metabolic activity of MTX and FA-treated organoids at different stages of treatment. MTX-treated organoids were supplemented with FA at different time points (0, 24, 48 and 72 h) with 5 µg/mL FA (n = 4). Metabolic activity was measured by WST-1 assay. All data are presented as Mean ± SEM. ####p < 0.0001 represents the significant differences observed between untreated organoids and MTX-treated organoids. ^^^p < 0.001 represents the significant differences between FA-treated organoids and MTX-treated organoids. **p < 0.01 and ****p < 0.0001 represent the differences observed between MTX-treated organoids and MTX/FA-treated organoids.

Figure 5

Cytokine and chemokine production in the MTX-induced mucositis organoids. Organoids were treated with 10, 100 and 1000 ng/mL MTX and incubated for 96 h. At different time points supernatant was collected and CXCL10 (A), CCL5 (B), TNF-α (C) and IL-1β (D) measured by Multiplex Immunoassay (n = 4). All data are presented as mean ± SEM. * (p < 0.05), ** (p < 0.01) and *** (p < 0.001) represent the significant changes between the MTX-treated and untreated groups.

Figure 6

Relative metabolic activity of MTX-treated organoids after butyrate, propionate and acetate administration. Organoids were treated with MTX and different doses of (A) butyrate (0.25–1 mM), (B) propionate (0.25-4 mM) and (C) Acetate (0.25-8 mM) for 96 h. Control groups were treated with different doses of SCFAs (n = 4–7). Relative metabolic activity was measured by WST-1. All data are presented as Mean ± SEM. ####p < 0.0001 represents the significant differences observed between untreated organoids and MTX-treated organoids. *p < 0.05, **p < 0.01 and ***p < 0.001 and ****p < 0.0001 represent the differences observed between MTX-treated organoids and MTX/SCFAs-treated organoids.