Human ALL blasts express high levels of OPN when localized to the endosteal niche, express OPN receptors, and adhere to tcOPN in vitro. (A) Representative micrographs of OPN IHC analysis of BM biopsies from control patients and patients with pre-B ALL. Leukemic lymphoblasts were found to express OPN in 6 of 6 ALL cases examined, and representative images are shown of 3 ALL cases and 1 control case. A gradient of OPN expression was detected in the leukemic marrows, with high OPN-expressing blasts localized near the trabecular bone surfaces. (B) Flow cytometry analysis of 6 primary ALL samples demonstrating variable expression of known OPN receptors. (C) In vitro adhesion assay to tcOPN of the 6 primary ALL samples from (B) demonstrating significant adhesion to OPN of blasts isolated from patients 1 to 3. (D) Intravital confocal imaging of the calvarial marrow of mice engrafted with DiR-labeled primary ALL cells (ALL #3 from Figure 1B-C). Mice were engrafted 42 days before imaging. Dormant DiR-retaining cells are represented in green; extracellular OPN is represented in red. The far right panel is a magnification of panel 3. A significant portion of dye-retaining blasts was found co-localized with or adjacent to areas of high OPN expression (n = 3).