OPN neutralization increases leukemia burden by decreasing the proportion of dormant tumor cells. (A) In vitro adhesion of Nalm-6 to thrombin-cleaved human and murine OPN is blocked by anti-OPN neutralizing antibodies, n = 3, *P = .0008, **P = .0014. OPN-coated dishes were pretreated with OPN-neutralizing antibodies before plating cells. (B) Nalm-6 homing in anti-OPN– vs isotype control-treated mice is equivalent. Intravital confocal microscopic study was used to examine the number of Nalm-6 leukemia cells in the calvarial BM of control vs anti-OPN–treated mice at 24 hours after engraftment; n = 3, *P = .5. (C) The antibody treatment scheme used in the OPN-neutralizing studies. (D-E) Flow cytometric quantification of GFP+ leukemia cells in BM aspirates from control and OPN-neutralized mice demonstrates increased disease burden in anti-OPN–treated mice. The paired analysis of the data is shown on the right; n = 7, *P = .02. (F) The proportion of Ki-67–positive leukemia cells is significantly higher in the BM of OPN-neutralized mice compared with isotype control-treated mice; n = 4, *P = .0005. (G) The percentage of F4/80-positive macrophages is equivalent in OPN-neutralized and isotype control-treated leukemic mouse BM; n = 4, *P = .5. (H) To determine whether OPN directly alters Nalm-6 proliferation, cells were incubated with tcOPN, an OPN neutralizing Ab, control immunoglobulin, or the chemotherapeutic agent SAHA. SAHA induced a dramatic decrease in proliferation, but neither incubation with tcOPN nor OPN-neutralizing Ab (to block autocrine binding of OPN secreted by Nalm-6 in culture) affected proliferation (n = 3). These data demonstrate that soluble OPN does not directly induce Nalm-6 dormancy.