Integrative Spatial Multi-Omics Reveal Niche-Specific Inflammatory Signaling and Differentiation Hierarchies in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a clonal disorder characterized by immature blasts and arrested differentiation that primarily affects the bone marrow (BM) and occasionally presents as extramedullary (EM) disease. EM manifestations highlight AML's adaptability to distinct microenvironments, which we examined using spatial analyses of medullary and EM tissues. We describe a workflow for Visium-based spatial transcriptomics in medullary and EM AML, revealing insights into cell-cell communication and the spatial organization of AML hierarchies. In BM, monocytes and granulocyte-monocyte progenitors colocalized with leukemic populations, sharing molecular signatures with those in EM sample. CXCL12-CXCR4-mediated communication correlated with PI3K/AKT/mTOR signaling in inflammatory niches. Trans-differentiation signals concentrated in AML-infiltrated regions; committed-like AML cells resided in inflammatory niches distant from trabeculae, while primitive-like cells localized near the endosteal niche. GeoMX digital spatial profiling and Opal multiplex fluorescent immunohistochemistry provided orthogonal validation. Overall, our study offers a valuable multimodal resource for exploring AML spatial biology with potential applications in other BM malignancies. Overall design: Bone Marrow (BM) and Extramedularry (EM) tissue biopsies were routinely collected before treatment initiation from 2 patients with newly diagnosed AML. The samples were formalin fixed, and BM samples were decalcified using 10% formic acid. Following fixation and decalcification, the samples were embedded in paraffin and stored at room temperature before they were cut into 4-µm sections. Visium Spatial Gene Expression (Direct placement; v1) and Visium CytAssist Spatial Gene Expression (CytAssist-enabled; v2) protocols apllied on each BM and EM samples. Libraries sequenced using Illumina NovaSeq 6000.