High-dimensional spatiotemporal single-cell atlas and 3D imaging of the bone marrow microenvironment during CML...

The bone marrow microenvironment (BMME) is essential for hematopoiesis and immunity, yet spatiotemporal single-cell analysis during leukemogenesis remains challenging. We characterized the BMME in femurs from wild-type and chronic myeloid leukemia (CML) mice at 7, 14, and 21 days after induction by highly multiplexed and 3-dimensional (3D) microscopy. Using a 54-marker codetection by indexing panel, we profiled 2 033 725 cells in 55 regions of interest and identified 41 cell types. During CML progression, we observed myeloid and progenitor cell expansion, increased programmed death ligand 1-positive leukemic cells, programmed death 1 (PD-1) upregulation on CD4+ and CD8+ T cells, and a profound loss of B cells, plasma cells, and bone cells. Advanced CML exhibited a striking expansion of immature, pericyte-deficient vasculature that disrupted vascular niches and impaired hematopoietic stem/progenitor cell positioning. Spatial mapping revealed leukemia-specific cellular neighborhoods enriched in PD-1+CD8+ T cells, suggesting localized immune exhaustion. Early CML showed increased contacts between plasmacytoid dendritic cells and megakaryocytes (MKs), whereas advanced CML featured heightened MK emperipolesis of nonleukemic granulocytes. MKs were morphologically irregular in CML mice and patient bone marrow biopsies. In contrast, in mice with acute myeloid leukemia, vasculature and MKs were reduced, whereas the remaining MKs retained normal morphology. Laser-capture microdissected MKs from patients with newly diagnosed CML had reduced cytoskeleton gene expression, which was reversed in advanced cases treated with tyrosine kinase inhibitors. 3D imaging revealed vascular disorganization and depleted MKs in the diaphysis, underscoring region-specific pathology. Together, this study provides a spatiotemporal single-cell atlas of the BMME during leukemic progression, showing how leukemic cells reprogram it to support their expansion and immune evasion.