CO52 | Deciphering the crosstalk between stroma and acute myeloid leukemia cells: an in vitro model to uncover the mechanisms underlying extramedullary progression in acute myeloid leukemia

Acute myeloid leukemia (AML) may present with extramedullary localizations (EM), in which leukemic blasts infiltrate anatomical districts other than the bone marrow (BM) or peripheral blood (PB). Changes in leukemic cells and in the tumor microenvironment (TME) may support this phenomenon. We previously demonstrated that the “metastatic” dissemination linked to EM involvement is triggered by the activation of the epithelial-mesenchymal transition (EMT) program and it is supported by the capability of blasts to digest the extracellular matrix (ECM), thus promoting their motility. We now aim at evaluating the contribution of the tumor microenvironment, particularly mesenchymal stromal cells (MSCs), in enhancing the metastatic potential of leukemic cells in vitro. We successfully established direct co-cultures between OCI-AML3 cells and primary MSCs freshly isolated from AML patients (n=3) and healthy donors (HDs, n=3). Leukemic cells ± 20 mM metformin, which we showed to strongly reduce their migration and invasion potential, were plated on the MSC feeder layer once they reached approximately 80% confluence. At the stopping point of the experiment (48h), Boyden Chamber assays were performed to assess the proportion of transmigrated cells. To study the functional interactions of MSCs and OCI-AML3, we used an 8-well chamber slide where we plated the cells previously labeled with DiD and DiI lipophilic tracers, and we subsequent observed them at confocal microscopy. Co-cultures of MSCs from AMLs and HDs revealed that MSCs boost OCI-AML3s migration and, in the case of AML-MSCs, also invasion (P