Myeloid Differentiation in Myelodysplastic Neoplasms and Leukemic Cells: Role of Azacitidine and Direct Contact with Mesenchymal Stromal Cells

Background. Ineffective hematopoiesis in the bone marrow (BM) is one of the key characteristics of myelodysplastic neoplasms (MDS), that are currently treated with Azacitidine. The BM microenvironment plays a pivotal role possibly affecting cell proliferation and differentiation, as mesenchymal stromal cells (MSCs) are believed to regulate hematopoietic cell behavior, potentially affecting MDS leukemic progression (Pontikoglou CG, Matheakakis A and Papadaki HA, 2023). Therefore, this study aims to further investigate the hematopoiesis process in MDS, focusing on the interactions between MSCs and hematopoietic cells, to elucidate the importance of their cell-cell direct contact, and exploring the possible role of Azacitidine treatment in the hematopoiesis mechanism.

Methods. 10 bone marrow and peripheral blood samples from patients with higher-risk MDS (IPSS-R intermediate, high, or very high), recruited at the IRCCS-Hematology Institute “L e A Seràgnoli” according to the Declaration of Helsinki, were collected at baseline and during therapy with Azacitidine. Flow cytometric phenotypic analyses of these samples were carried out using a panel of 15 markers including CD11b and CD14. In addition, in vitro direct contact and transwell co-cultures were set up using MSCs obtained from different sources (HS-5 cell line and dental pulp-derived MSCs) and hematopoietic cells (KG-1/THP-1 cell lines and MDS primary samples), while co-cultures between healthy MNCs and MDS-MSCs are under analysis. In our in vitro co-culture model, cellular behavior was assessed via scanning and transmission electron microscopy (SEM and TEM), as well as live microscopy; moreover, the expression of CD11b and CD14 was quantified by flow cytometric analyses before and after co-culture, and before and after pharmacological treatment. All analyses were carried out in suspension cells and in cells adherent to MSCs, using CD73 marker to distinguish adherent cell populations at the flow cytometer.

Results. CD11b and CD14 markers increased after Azacitidine treatment in MDS responder patients, while they decreased in non-responders. This led us to divide MDS samples in CD11b high/low and CD14 high/low, to be used for ongoing co-culture experiments. Our co-culture in vitro model revealed that MSCs may stimulate cell adhesion and induce leukemic cells to divide in two sub-populations: suspended and adherent to MSCs. Moreover, during co-culture, a high percentage of CD34+ KG-1 cells at first adhered to MSCs, followed by a progressive CD34+ decrease. At the same time, flow cytometric analyses revealed a statistically significant increase in CD11b and CD14 expression in both suspended and MSCs-adherent KG-1 populations after 144h of co-culture, compared to control KG-1 cells alone. This behavior was detected even in THP-1 cells, suggesting that MSCs may indeed affect myeloid differentiation. Additionally, THP-1 cells already induced to macrophage differentiation by phorbol myristate acetate, confirmed that already differentiated THP-1 cells are less adherent to MSCs. Furthermore, transwell experiments showed that CD11b and CD14 markers increased in KG-1 and THP-1 co-cultured cells as well, but in this case the increase was not statistically significant compared to the control. As for morphology studies, SEM observations revealed increased vesicles secretion at later stages of co-culture, suggesting their possible involvement in the cross-talk between the two cellular sub-populations, to be confirmed by ongoing immunolabeling experiments.

Conclusions. All in all, our findings show that Azacitidine response is associated with myeloid differentiation in MDS cells and that these processes may also depend on MSCs. Indeed, our in vitro model showed that myeloid differentiation could be triggered by factors secreted by MSCs, as confirmed by transwell experiments, and further amplified by cell-to-cell direct contact, that, as showed by direct contact co-cultures, seems to be essential for myeloid differentiation. Ongoing analyses are trying to elucidate the effect of MSCs on MDS during myeloid differentiation or leukemic progression, possibly leading to novel MDS therapeutic strategies based on myeloid-derived suppressor cells or BM microenvironment targeting.

Curti:Abbvie: Honoraria; Menarini stemline: Honoraria; Jazz Pharmaceutics: Honoraria; Pfizer: Honoraria, Research Funding. Finelli:Celgene BMS: Consultancy; Novartis: Consultancy; Takeda: Consultancy.