Fig. 1.
F-HA binding to BCa cell lines is heterogeneous and linked to malignant phenotype. (A) Schematic for fluorescent labeling of HA with hydrazide functionalized A647 or TR dyes. (B) HA-binding profiles measured by FACS after addition of A647-HA (+F-HA) to cells for 45 min at 4 °C (n = 3–5). Positive binding was determined by FACS geometric mean values of populations within peak 2, and the gray dashed line represents background fluorescent level of intact cells before addition of F-HA (−F-HA), which was similar for all cell types. All F-HA–binding profiles span from 0 to 105 fluorescent signals. The graphs indicate heterogeneity indices of the profiles as measured by Het.I = median × (AUC2/AUC1). (C and D) Comparison of MDA-MB-231 (basal-like) and SKBR-3 (luminal) HA-binding levels from 2 to 120 min after addition of F-HA to cells grown in 2D and 3D laminin-rich gels, respectively. n ≥ 3. (Scale bar: 50 µm.) Error bars represent SD and normalized SD, respectively. (Upper and Lower Left in C and D) Optical and scanning electron micrographs of MDA-MB-231 and SKBR-3 cells in 2D and 3D culture before F-HA binding. F-HA–binding profiles and morphology of MDA-MB-231 cells before (E, Left) and after reversion (E, Right) (57) to a more nonmalignant phenotype. n = 3. (Scale bar: 80 µm.)