A high-dimensional spatiotemporal single-cell and 3D atlas of the bone marrow microenvironment during CML progression: Leukemia-driven vascular remodeling, immune dysregulation, and megakaryocyte abnormalities

Abstract

Abstract The bone marrow microenvironment (BMME) critically regulates hematopoiesis and immune function. Disruptions in its cellular composition and spatial architecture drives disease progression, particularly in leukemia. As a semi-solid tissue enclosed in bone, it is technically challenging to analyze in situ. Yet, high-dimensional, spatially resolved single-cell data are crucial for understanding chronic myeloid leukemia (CML) progression. We established a protocol for CO-Detection by indEXing (CODEX) multiplexed imaging on fixed-decalcified-frozen BM that preserves tissue integrity and native marrow architecture to chart BMME changes during CML evolution. Using an immunocompetent, aggressive, BCR::ABL1-driven CML mouse model, we profiled femoral sections from wild-type and CML mice 7, 14 and 21 days post-induction with a 54-marker CODEX panel and full-thickness 3D light-sheet microscopy focused on megakaryocytes and the vascular network. Using CODEX, we imaged and annotated 2,033,725 cells across 55 regions of interest. An unsupervised clustering pipeline, iteratively refined by supervised annotation and image-based validation, resolved 41 cell types spanning major hematopoietic lineages and rare stromal populations. During CML progression, PD-L1+ leukemic and antigen presenting cells expanded markedly, with significant increase in myeloid, vascular, and progenitor compartments (proerythroblasts, early neutrophil progenitors, common monocyte progenitors and megakaryocyte progenitors). Conversely, B cells, plasma cells and osteolineage cells declined sharply. Despite stable total T-cell abundance, both CD8+ and CD4+T cells were contracted, while PD-1+CD8+ and PD-1+CD4+ subsets expended significantly. Cellular neighborhood (CN) analysis identified 14 distinct CNs, representing major BM functional regions (e.g., vascular, B cell, erythroid, bone, and leukemia niches). Notably, PD-1+CD8+ T cells were mostly enriched within the leukemia niche in advanced CML. Advanced CML also exhibited a striking expansion of immature, pericyte-deficient vasculature that disrupted vascular niches and impaired hematopoietic stem/progenitor cell (HSPC) positioning. Advanced CML HSPCs were enlarged in size, which is associated with loss of quiescence and functional decline. Additionally, early-stage CML showed increased plasmacytoid dendritic cell and megakaryocyte contacts, whereas advanced CML exhibited increased emperipolesis of non-leukemic granulocytes within megakaryocytes. Megakaryocytes were morphologically irregular in CML mice and BM trephine biopsies from CML patients. Laser-capture microdissected megakaryocytes from newly diagnosed patients had reduced expression of cytoskeleton genes, which was reversed in advanced cases treated with tyrosine kinase inhibitors. 3D light-sheet imaging revealed vascular disorganization and depleted megakaryocytes in the diaphysis, underscoring region-specific pathology. Here, we present a detailed spatiotemporal single-cell and 3D atlas of the BMME during CML progression. Our findings reveal how leukemic cells actively remodel the BMME, establishing specialized niches that not only support their expansion but also disrupt normal hematopoiesis and suppress antitumor immunity. By characterizing these dynamic alterations at single-cell resolution, we provide critical insights into the mechanisms driving CML progression and immune evasion, offering potential avenues for therapeutic intervention.