Dormant Nalm-6 GFP localize to areas of high OPN expression. (A) Nalm-6 GFP were labeled with DiR (green), a lipophilic membrane dye that is retained by noncycling cells. At 42 days after engraftment, mice were injected with AF647-labeled isotype control or anti-OPN (red) antibodies to visualize extracellular OPN. The calvarial BM was imaged using intravital confocal microscopic study ∼20 hours after antibody injection. Cycling Nalm-6 GFP cells that have diluted the membrane dye to undetectable levels are represented in blue. (B) Proportion of dye-retaining (dormant) and dye-depleted (cycling) leukemia cells that co-localize with BM extracellular OPN, *P < .0001. Whereas few proliferating Nalm-6 GFP are directly adjacent to extracellular OPN, the majority of dormant Nalm-6 GFP co-localize with the OPN signal, suggesting a functional relationship between stromal OPN and ALL dormancy.