The tumor microenvironment contains increased amounts of both tumor and host OPN. (A) RT-PCR shows hOPN mRNA transcripts in tumor-engrafted mice. Human RPL13A and pan-species β actin housekeeping genes serve as loading controls. (B) Quantitative PCR analysis for murine OPN transcripts shows increased levels in Nalm-6 GFP mice, *P = .001, **P = .007. (C) IHC analysis of OPN expression in femurs from an OPN knockout mouse, a control nonengrafted SCID mouse and a SCID mouse with diffuse Nalm-6 GFP leukemic BM infiltration. OPN is detected at the endosteal surface in control mice. In tumor mice, OPN+ leukemic cells are localized near the endosteal niche (arrows). (D) ELISA analysis demonstrates hOPN in BM serum from leukemic mice; control n = 2, tumor n = 6, *P < .0001. (E-F) Intravital confocal microscopic examination of extracellular OPN (red) expression in control SCID mouse calvarial BM. (G-H) Representative images of intravital confocal microscopic study of extracellular OPN expression in the calvarial BM of Nalm-6 GFP (green)–engrafted SCID mice at D35 (G) and D42 (H). Control images E and F correspond to the regions imaged in panel G and H, respectively. These results indicate that extracellular OPN levels are most intense where tumor burden abuts bony margins. Scale bars equal 50 µm.