EXPLORATION OF THE TUMOR MICROENVIRONMENT: DECODING SECRETOME- INDUCED HEMATOPOIESIS DISRUPTION AND IMMUNOSUPPRESSION IN A 3D MODEL OF ACUTE MYELOID LEUKEMIA

Acute myeloid leukemia (AML) is a hematological disease that accounts for 20% of all the pediatric acute leukemias, and primary treatment is chemotherapy. Despite AML outcome has significantly improved over the past 20 years, it still represents a challenge due to the considerable occurrence of relapse, which is the main cause of treatment failure. Thus, there is still an urgent need for novel therapeutic strategies to achieve better outcomes in pediatric AML. The BM niche plays a pivotal role in preserving the homeostasis and functionality of hematopoietic stem cells (HSCs). However, during the onset and progression of AML, this microenvironment undergoes extensive remodeling, evolving into a niche that fosters leukemic growth. Mesenchymal stromal cells (MSCs), a critical non-hematopoietic cell population within the BM, contribute to AML cell proliferation while impairing normal hematopoiesis through mechanisms such as the secretion of soluble factors. Nevertheless, the precise role of MSCs in AML pathophysiology remains incompletely understood, underscoring the need for deeper investigation. We previously developed an advanced 3D model of BM niche, consisting of a porous scaffold of Hydroxyapatite and Collagen type I, where MSCs are seeded. After one week, primary AML blasts are introduced into the system. This model supports the long-term culture of AML blasts for up to 21 days. We demonstrated that MSCs isolated from pediatric AML patients at diagnosis (AML-MSCs) exhibit distinct transcriptional and functional profiles compared to MSCs derived from age-matched healthy donors (h-MSCs). These differences also result in significant alteration in the secretome profile, suggesting a potential role in disease pathogenesis and microenvironmental remodeling. During my PhD, I focused on the role of the altered AML-MSCs secretome within the leukemic niche, specifically its impact on hematopoietic dysfunction and immunosuppression. Additionally, to develop a more predictive model of AML niche, I enhanced our 3D system by incorporating other cell types that are known to play critical roles in the BM. Experiments started using liquid chromatography-mass spectrometry (LC-MS) analysis on a series of 3D-cultures set up to mimic the AML or the healthy niche to identify the MSCs contribution. By LC-MS of the culture media we identified 15 differentially and newly secreted proteins between the 7 days 3D cultures of AML-MSCs+AML blasts and h-MSCs+AML blasts. Then, 3 of these factors emerged as candidates playing a role in the AML niche and were further investigated. Here, we demonstrated that these 3 factors are involved in supporting AML blasts proliferation but also influence HSCs fate by reducing their proliferation and supporting the maintenance of stemness. Then, by utilizing iPSCs (induced Pluripotent Stem Cells), we also explored the role of the 3 AML factors during hematopoietic processes, observing that AML factors can imprint HSCs to differentiate towards the monocyte and macrophage lineages. We underpinned that these factors polarize macrophages into an M2-like phenotype to induce Tumor-Associated Macrophages (TAMs) which play a fundamental role in the tumor microenvironment (TME), particularly by inducing an immunosuppressive niche as shown by a reduced T cell proliferation and activation when introduced in the 3D system. These findings highlight that the 3 AML factors have the potential of modifying the niche and should be further evaluated as an additional therapeutic strategy. The relevance of immune surveillance in the context of leukemia treatment pushed to the generation of models that could help in dissecting how cells interact and modify their function, and the generation of an enhanced 3D model including additional monocytes, endothelial and neuronal cell types is pivotal for future more effective treatments that not only target leukemic cells but also reestablish a functional healthy BM microenvironment.