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Patient-derived oral mucosa organoids as an in vitro model for methotrexate induced toxicity in pediatric acute lymphoblastic leukemia.

PloS one (2020-05-19)
E Driehuis, N Oosterom, S G Heil, I B Muller, M Lin, S Kolders, G Jansen, R de Jonge, R Pieters, H Clevers, M M van den Heuvel-Eibrink
ABSTRACT

We have recently established a protocol to grow wildtype human oral mucosa organoids. These three-dimensional structures can be maintained in culture long-term, do not require immortalization, and recapitulate the multilayered composition of the epithelial lining of the oral mucosa. Here, we validate the use of this model to study the effect of Leucovorin (LV) on Methotrexate (MTX)-induced toxicity. MTX is a chemotherapeutic agent used in the treatment of pediatric acute lymphoblastic leukemia. Although effective, the use of MTX often results in severe side-effects, including oral mucositis, which is characterized by epithelial cell death. Here, we show that organoids are sensitive to MTX, and that the addition of LV reduces MTX toxicity, in both a concentration- and timing-dependent manner. Additionally, we show that a 24 hour 'pretreatment' with LV reduces MTX-induced cell death, suggesting that such a pretreatment could decrease mucositis in patients. Taken together, we provide the first in vitro model to study the effect of MTX on wildtype oral mucosa cells. Our findings underscore the relevance of the clinically applied LV regimen and highlight the potential of this model to further optimize modifications in dosing and timing of Leucovorin on oral mucosa cells.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Perchloric acid, ACS reagent, 70%
Sigma-Aldrich
Dispase® II, protease
Sigma-Aldrich
Trypsin from bovine pancreas, TPCK Treated, essentially salt-free, lyophilized powder, ≥10,000 BAEE units/mg protein
Sigma-Aldrich
CHIR99021, ≥98% (HPLC)
Sigma-Aldrich
Nicotinamide, BioReagent, suitable for cell culture, suitable for insect cell culture
Sigma-Aldrich
Folinic acid calcium salt hydrate, BioXtra, ≥99.0% (HPLC)
Methotrexate, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
L-Glutamic acid-15N, 98 atom % 15N