Fig. 2. E-selectin regulates entry of BCCs into bone from peripheral circulation.

(A) Cell surface expression of CXCR4 versus day 1 BCC BM homing (R² = 0.0013, linear regression; n = 3 per cell line). (B) BCC BM homing in mice treated ± CXCR4 inhibitor AMD3100 (n = 3 each cell line). NS, not significant. (C) BCC BM homing in mice treated ± CXCR4 neutralizing antibodies (MCF-7: n = 3; 1833: n = 4). (D) BCC BM homing in mice treated ± pertussis toxin to broadly inhibit chemokine signaling before engraftment (n = 3 each cell line). (E) E-selectin expression (green) in the sinusoidal regions of calvarial BM was detected by in vivo microscopy using anti–E-selectin–Cy5 antibodies and was colocalized with fluorescently labeled BCCs (cyan) imaged simultaneously on day 1 after intracardiac engraftment (dextran-FITC, red; montage contains multiple images). (F) BCC homing was quantified in mice treated ± E-selectin inhibitor GMI-1271 at both initial (2 hours; MCF-7: **P = 0.0068, n = 3; MDA-MB-231: *P = 0.0356, n = 3; unpaired, two-way t test) and late (20 hours; MCF-7: **P = 0.0052, n = 3; MDA-MB-231: *P = 0.0491, n = 3; unpaired, two-way t test) time points. Representative images shown (BCC, green; dextran-FITC, red; montage contains multiple images). (G) Schematic of MCF-7 stem (CD44⁺CD24^−/low) and non-stem (CD44⁺CD24^−/high) cell isolation by flow sorting and engraftment ± GMI-1271 into female severe combined immunodeficient (SCID) mice. SSC, side scatter; FSC, forward scatter. (H) BM homing of MCF-7 stem and non-stem cell populations ± GMI-1271 at 20 hours after engraftment (**P = 0.0026, n = 3; unpaired, two-way t test). Scale bars, 100 μm.