Osteopontin-c expression levels modulate leukemic cell morphological plasticity and transmigration across a blood-brain barrier-on-a-chip model

Central nervous system infiltration remains a major clinical challenge in B-cell precursor acute lymphoblastic leukemia, particularly in high-risk subgroups such as KMT2A-rearranged cases. Osteopontin, especially its splice variant Osteopontin-c (OPNc), has been associated with aggressive disease features, including central nervous system involvement, although its functional role in leukemic dissemination remains unclear. Here, we developed a multicellular human blood-brain barrier-on-a-chip model to investigate the contribution of OPNc to leukemic cell transmigration. The microfluidic system recapitulates key structural and functional features of the blood-brain barrier (BBB), including endothelial, pericyte, and astrocyte interactions, and exhibits selective barrier properties. Using this system, we demonstrated that RS4;11 leukemic cells adhere to, infiltrate, and transmigrate across the BBB. Silencing of OPNc significantly reduced leukemic cell migration and invasion in transwell assays and was associated with altered cellular morphology, suggesting impaired migratory plasticity. OPNc-silenced cells showed consistent trends toward reduced infiltration and increased circularity rates. Altogether, these findings evidence a role for OPNc as one of the mediators of leukemic cell behavior associated with CNS infiltration and establishing the BBB-on-a-chip as a physiologically relevant platform for studying leukemic dissemination. Teaser abstract: Central nervous system (CNS) infiltration remains a major challenge in high-risk B-cell precursor acute lymphoblastic leukemia. Using a multicellular human blood-brain barrier-on-a-chip, we demonstrate that RS4;11 leukemic cells adhere to and transmigrate across the barrier. OPNc silencing reduced migration and invasion and altered cellular morphology, suggesting impaired migratory plasticity. These findings support OPNc as one of the mediators of leukemic behavior associated with CNS infiltration and establish the BBB-on-a-chip as a physiologically relevant platform to investigate leukemic dissemination.