High-Dimensional Spatiotemporal Single-Cell Atlas and 3D imaging of Bone Marrow Microenvironment during CML Progression

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 post-induction by highly multiplexed and 3D microscopy. Using a 54-marker CODEX 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 PD-L1+ leukemic cells, 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, whereas advanced CML featured heightened megakaryocyte emperipolesis of non-leukemic granulocytes. Megakaryocytes were morphologically irregular in CML mice and patient BM biopsies. In contrast, in mice with acute myeloid leukemia, vasculature and megakaryocytes were reduced, while remaining megakaryocytes retained normal morphology. Laser-capture microdissected megakaryocytes from newly diagnosed CML patients had reduced cytoskeleton gene expression, which was reversed in advanced cases treated with tyrosine kinase inhibitors. 3D imaging revealed vascular disorganization and depleted megakaryocytes 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.