Processed single-cell RNA-seq from the study 10X Genomics CellRanger output (barcodes.tsv, genes.tsv, matrix.mtx) for each each sample Metadata Distinct Stromal Cell Populations Define the B-cell Acute Lymphoblastic Leukemia Microenvironment Mauricio N. Ferrao Blanco1, Bexultan Kazybay1, Mirjam Belderbos1, Olaf Heidenreich1, Hermann Josef Vormoor1,2 1 Princess Máxima Center for Pediatric Oncology, Utrecht, the Netherlands 2 University Medical Center Utrecht, Utrecht, the Netherlands Abstract The bone marrow microenvironment is critical for B-cell acute lymphoblastic leukemia (B-ALL) but its cellular heterogeneity remains poorly defined. Here, we employed single-cell RNA sequencing to comprehensively characterize the stromal and hematopoietic niches in pediatric B-ALL. Our analysis revealed two distinct mesenchymal stromal cell (MSC) populations as primary leukemia-supportive niches: early mesenchymal progenitors and adipogenic progenitors. Single-cell transcriptomic analysis infers that ALL blasts use distinct cell-cell interactions to communicate with the different stromal populations. Purified adipogenic progenitors from the bone of children with ALL support survival of the leukemic blasts ex vivo and their signature is enriched in relapse samples. Our data establish adipogenic progenitors as a distinct and novel component of the ALL niche.