Distinct Stromal Cell Populations Define the B-cell Acute Lymphoblastic Leukemia Microenvironment

Processed single-cell RNA-seq from the study 10X Genomics CellRanger output (barcodes.tsv, genes.tsv, matrix.mtx) for each each sample Metadata Seurat object of the integrated scRNAseq dataset Xenium object of the spatial transcriptomic data 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 plays a critical role in B-cell acute lymphoblastic leukemia (B-ALL) progression, yet its cellular heterogeneity remains poorly understood. Using single-cell RNA sequencing on patient-derived of bone marrow aspirates from pediatric B-ALL patients, we identified two distinct mesenchymal stromal cell (MSC) populations: early mesenchymal progenitors and adipogenic progenitors. Spatial transcriptomic analysis further revealed the localization of these cell types and identified a third stromal population, osteogenic-lineage cells, exclusively present in the bone biopsy. Functional ex vivo assays using sorted stromal populations derived from B-ALL patient bone marrow aspirates demonstrated that both early mesenchymal and adipogenic progenitors secrete key niche-supportive factors, including CXCL12 and Osteopontin, and support leukemic cell survival and chemoresistance. Transcriptomic profiling revealed that B-ALL cells interact differently with stromal subtypes. Notably, adipogenic progenitors, but not early mesenchymal progenitors, provide support to leukemic cells through interleukin-7 and VCAM1 signaling. Stromal cells from B-ALL patients exhibited an enhanced adipogenic differentiation capacity compared to healthy controls. Moreover, co-culture experiments showed that B-ALL cells induce adipogenic differentiation in healthy MSCs through a cell contact-dependent mechanism. Adipogenic progenitors were also enriched in relapse samples, implicating them in disease progression. These findings highlight the complexity of the B-ALL microenvironment and identify different specialized stromal niches with which the leukemic cells can engage.