Abstract:

Bone marrow (BM) is a complex tissue where hematopoiesis, the process of blood cell formation, takes place during adult life. Genetic alterations in Hematopoietic Stem Cells (HSC) can increase the risk of uncontrolled proliferation and halted differentiation of these immature cells, leading to development of aggressive blood cancers such as acute leukemias. These diseases affect both children and adults and often have a poor prognosis. T-cell acute lymphoblastic leukemia (T-ALL), which originates from malignant T-cell precursors, is particularly invasive. While high-dose chemotherapy is the primary treatment, it is commonly associated with resistance, relapse, and significant side effects. Recent research highlights that acute leukemias not only have a genetic basis but also impact the BM microenvironment (BMM). The BMM, composed of non-hematopoietic stromal cells and acellular components, plays a crucial role in supporting hematopoiesis in both physiological and pathophysiological conditions. This literature search, summarized in a review in revision in Leukemia journal and included in the appendix of the thesis, highlights how the BMM, particularly the vasculature, provides critical survival signals to leukemic cells, undergoing significant remodeling during leukemia progression.

The objective of my thesis is to explore the malignant adaptation of the BMM to T-ALL progression, with the aim of implementing effective molecular strategies targeting this interaction to improve patient survival.

In the first part of my work, we sought to identify key players in the cross-talk between endothelial cells (EC) and T-ALL cells by analyzing the BM vascular niche during leukemia progression. To do so, we used T-ALL mouse models in association with fluorescent reporters in the BMM. We combined multicolor spectral cytometry and microscopy to establish a panel of surface and extracellular matrix (ECM) proteins, which could reveal vascular niche heterogeneity and its changes associated with T-ALL. We observed global changes in the BM stromal population, particularly the unbalanced distribution of osteoprogenitors, pericytes, and BMEC in the BM of T-ALL mice at the late stage of the disease. By analyzing the T-ALL microenvironment at different stages (early, intermediate, and late) of disease progression, we identified 9 distinct BMEC subpopulations, of which 3 were notably altered and emerged as novel therapeutic targets. Our data revealed an important loss of sinusoidal vessels (VEGFR-2+; Vcam-1+) and an expansion of capillaries with an arterial phenotype associated with the expression of specific integrins. Spatially, we observed a disorganized vasculature showing loss of the sinusoidal markers CD105 and increased expression of markers associated with arterioles (Sca-1high; Laminin+). Additionally, we identified the progressive replacement of sinusoidal vessels by transitional capillaries, confirming the drastic structural and functional changes in the BM vasculature during T-ALL progression.

In the second part of my work, we aimed to validate the involvement of specific BMM mediators in leukemia progression. To do so, we worked on the establishment of novel preclinical strategies to disrupt the crosstalk between BMM and T-ALL. On one hand, we used mouse models to set up genetic engineering strategies to specifically modulate gene expression in BMEC within the in vivo BMM. On the other hand, we used a novel model of vascularized BM-on-chip developed in our lab, incorporating human EC which we targeted with shRNA against selected candidates. These systems would allow for high throughput screening of multiple targets within a relatively short time frame and with reduced or no use of animals.

In conclusion, my thesis highlights a critical remodeling of the BM vascular niche during T-ALL progression, identifying specific endothelial subpopulations as novel therapeutic targets and setting the ground for innovative preclinical strategies to disrupt leukemia crosstalk with the BMM, ultimately aiming to improve patient outcomes.

Keywords: Leukemia; Bone marrow; T-ALL; T-cell; Cancer; Endothelial cells; Niche; Microenvironment; Vascular.