Macrophage infiltration into tumor spheroids.

(A) 7.5 x 103 MCF7 cells were seeded in agarose-coated 96-well plates to form three-dimensional spheroids. Picture is representative for 5 days old MCF7 tumor spheroids. (B) 7.5 x 104 CD14+ cells were added to 5 days old spheroids. Cellular composition of the spheroids subsequently cultured for 2 days in the absence (left panel) or presence (right panel) of CD14+ cells was determined by FACS analysis of EpCAM+ tumor cells and CD45+ immune cells. Graphs are representative for 3 independent experiments. (C) MΦ infiltration was determined as the proportion of CD45+ cells relative to all cells and is represented as mean ± SEM (n = 3). (D) CFSE-labeled CD14+ cells were added to 5 days old spheroids and co-cultured for 2 days. Infiltration was visualized via fluorescence microscopy.