Mapping the Spatial Proteome of Leukemia Cells Undergoing Fludarabine Treatment

Recent advancements in spatial biology have revolutionized our understanding of the organization and functional dynamics of cells and tissues. In this study, we applied Molecular Pixelation (MPX), a single-cell spatial proteomics assay, to investigate the modulation of the cell surface proteome in an in vitro drug screening model using the ETV6::RUNX1 acute lymphoblastic leukemia (ALL) cell line, Reh . Specifically, we focused on the in vitro response to fludarabine, a chemotherapeutic agent used prior to allogenic stem cell transplantation and chimeric antigen receptor (CAR)-T cell therapy in high-risk, refractory, or relapsed ALL patients. Using MPX, we quantified changes in protein abundance, spatial distribution, and colocalization of 76 targeted cell surface proteins in Reh cells before and after fludarabine treatment. Our analysis revealed 25 proteins with altered abundance, 24 proteins with increased polarity, and 138 protein pairs with modified colocalization following treatment. Notably, the tetraspanins CD82 and CD53, which are known for their roles in chemotherapy resistance, exhibited increased abundance, polarization, and colocalization post-treatment, suggesting their potential as a therapeutic scaffold. These findings underscore the unique ability of spatially resolved single-cell proteomics to uncover nuanced cellular responses that would otherwise remain undetected.