3-D tumor spheroid culture further increases the interactions between BCa cells and E-selectin coated surfaces by upregulating CD44V4 and sLe<sup>x</sup> expression.

<p>(a) Micrographs of MDA-MB-231 cells cultured in 2-D monolayer (left) and 3-D spheroid on PDMS (right). (b) Flow cytometry results of CD44V4 (left) and sLe<sup>x</sup> (right) expression on untreated (2-D monolayer grown) and tumor spheroid cultured MDA-MB-231 cells. Tumor spheroid cultured cells were treated with enzyme free dissociation buffer prior to experiments to obtain mostly single cell populations. Mean fluorescence intensity ratio of sample over isotype has been included. (c) Average rolling velocity (left) and average number of MDA-MB-231 cells found interacting with E-selectin coated surfaces (right) cultured in medium (untreated), 3-D spheroid, and spheroid conditioned medium. Bars represent mean ± SEM. Two-tailed unpaired student t-test was used. (d) CD44 mRNA expression of MDA-MB-231 cells that are cultured in control, IL-6, TNF-α, tumor spheroid, and plasma conditions, respectively, measured via qPCR.</p>