Fig. 1. BCCs hijack SDF-1⁺E-selectin⁺ vascular gateways to enter the bone.

(A) Discovery cohort of untreated, node-negative breast cancer patients revealed four subgroups based on gene expression. Of these, G3 had the highest recurrence-free survival, whereas G4 had an increased risk of late recurrence (≥5 years after diagnosis). In Analysis A, expression of 29 genes representing CXCR4, E-selectin ligands, and enzymes critical for posttranslational processing of E-selectin ligands was analyzed in a data set compiled from 46 microdissected samples (G3, n = 18; G4, n = 10). In Analysis B, these same 29 genes were analyzed in the ER⁺ subset of our discovery cohort of patients with untreated, node-negative disease [late recurrence (LR) ≥ 5 years, n = 66; recurrence-free (RF) > 15 years, n = 26]. LCM, laser capture microdissection. (B to D) The expression of 7 of 29 genes analyzed was significantly increased in microdissected tumor epithelium of patients with late recurrence (t test). In the ER⁺ subset of our discovery cohort, 3 of these 29 genes were significantly up-regulated in tumor samples from patients with late recurrence compared to those from patients with recurrence-free survival at ≥15 years (Mann-Whitney U test). (D) CXCR4 expression correlates with time to recurrence in the ER⁺ subgroup [***P = 0.00097, analysis of variance (ANOVA)] [P values presented in (B); red indicates P < 0.05]. (E) Intravital microscopy was used to image the calvarial BM of mice engrafted with fluorescent membrane dye–labeled BCCs in real time with single-cell resolution. Dextran-FITC (fluorescein isothiocyanate) was used to visualize the blood pool. As shown in the montage image of in vivo micrographs, sinusoidal vasculature, which is known to have a distinct SDF-1⁺E-selectin⁺ phenotype, is concentrated in distinct parasagittal locations (montage containing multiple images; scale bar, 200 μm). (F) Fluorescently labeled BCCs were engrafted by intracardiac injection in mice, and the calvarium was imaged in the initial 2 hours after engraftment. BCCs were detected entering the bone specifically in perisinusoidal areas. Cells were not detected in lateral or caudal regions that lack substantial sinusoidal vasculature (montage containing multiple images; scale bar, 100 μm).