Spatial multi-omics uncovers niche remodeling in acute myeloid leukemia
Acute myeloid leukemia (AML) is a heterogeneous malignancy with a poor prognosis, urgently requiring novel therapeutic options. The limited success of immunotherapies in AML has been mainly attributed to an immunosuppressive bone marrow (BM) microenvironment, where various immune and stromal cells have been implicated. However, the spatial organization and cellular interactions that underpin immune evasion within the AML BM arrow have remained poorly defined, because methods that rely on dissociated tissues lack spatial context and poorly capture various microenvironmental cell types.
To address this gap, we employed multi-omics single-cell spatial profiling to characterize the AML BM in situ. We constructed three tissue microarrays from formalin-fixed paraffin-embedded (FFPE) BM biopsies from 74 newly diagnosed pediatric and adult patients with various AML subtypes, and 24 non-leukemic controls. Spatial transcriptomics was performed using 10x Xenium with the pre-designed Human Multi-Tissue and Cancer panel, supplemented with probes for 100 genes to better characterize BM-resident cells, and probes for the RUNX1::RUNX1T1 fusion to aid identification of malignant cells (n=477 genes). Complementary spatial proteomics data were generated via imaging mass cytometry (IMC) using a 37-plex panel.
Combined single-cell analysis of more than 1.3 million high-quality cells enabled the spatial and phenotypic characterization of all major hematopoietic, stromal and endothelial lineages. Detection of the RUNX1::RUNX1T1 fusion directly confirmed malignant cell status in the subset of patients characterized by this translocation, with highest detection in immature, granulocytic, and eosinophil progenitor-like blasts, consistent with the bifurcated differentiation seen in this AML subtype (Derevyanko et al., bioRxiv 2025). Notably, both Xenium and IMC identified macrophages (median 2%) and stromal cells (median 4%) at substantially higher frequencies than in typical BM aspirate-based analyses. Further characterization of the stromal compartment (26,798 cells) revealed significant compositional differences between leukemic and non-leukemic BM, and identified a rare, AML-specific cell type expressing markers of endothelial-to-mesenchymal transition, previously described only in regenerating BM (Kenswil et al., Cell Stem Cell 2021).
Spatial transcriptome-based neighbourhood analysis identified 10 distinct cellular neighbourhoods (CNs), including three CNs shared between patients and controls (late myeloid; erythroid and B cells; and a diverse microenvironmental CN), while the other seven were AML-specific and correlated with molecular subtypes. IMC-based CN analysis supported these findings.
To further explore cellular crosstalk within the AML microenvironment, we performed spatially informed cell-cell communication analysis, which uncovered enhanced immunosuppressive interactions between blasts and their microenvironment, including the Galectin-9 – TIM-3 axis. Co-culture experiments confirmed upregulation of TIM3 in macrophages upon polarization towards an immunosuppressive phenotype by AML blasts.
In conclusion, we generated a comprehensive multi-omics single-cell spatial atlas of the AML BM, which provides insight into the AML microenvironment across molecular subtypes and ages, facilitating the identification of novel therapeutic targets and supporting the design of immunotherapeutic interventions in AML.