Long-term proliferation of immature hypoxia-dependent JMML cells supported by a 3D in vitro system

Abstract

Juvenile myelomonocytic leukemia (JMML) is a rare, clonal stem cell disorder occurring in early childhood and characterized by RAS pathway hyperactivation in 95% of patients. JMML is characterized by a hyperproliferation of granulocytes and monocytes, and little is known about the heterogeneous nature of leukemia-initiating cells, as well as of the cellular hierarchy of the JMML bone marrow (BM). In this study, we report on the generation and characterization of a novel patient-derived three-dimensional (3D) in vitro JMML model, called pd-JAO, sustaining long-term proliferation of JMML cells with stem cell features and patient-specific hallmarks. JMML cells brewed in the 3D model under different microenvironmental conditions acquired a proliferative and survival advantage when placed at low oxygen tension. Transcriptomic and microscopic analysis revealed the activation of specific metabolic energy pathways and the inactivation of processes leading to cell death. Furthermore, we demonstrated the pd-JAO derived cells' migratory, propagation and self-renewal capacities. Our study contributes to the development of a robust JMML 3D in vitro model to study and define the impact of microenvironment stimuli on JMML disease and the molecular mechanisms regulating JMML initiating and propagating cells. Pd-JAO may become a promising model for compound tests focusing on new therapeutic intervention aiming to eradicate JMML progenitors and control JMML disease.

O2-sensitive microcavity arrays: A new platform for oxygen measurements in 3D cell cultures

“…A large majority of experiments to date are performed under ambient atmosphere, which translates into hyperoxic culture conditions, or with extremely hypoxic values rather than with the tissue-typical oxygen concentrations ( Al-Ani et al, 2018 ; Tse et al, 2021 ). Moreover, typical tissue oxygen concentrations are sometimes not known because they can hardly be determined although they are essential for the maintenance of tissue-specific metabolism ( Carreau et al, 2011 ; Zhang et al, 2016 ; Cani et al, 2022 ). The measurement of oxygen gradients and also the regulation of the oxygen supply of the culture system is still a major challenge but will be necessary to mimic in vivo -like oxygen concentrations in the cell culture model.…”