Figure 1.

Morphology and behavior of cells during spheroid formation. (A–I) Twenty-four hour spheroid culture. Phase-contrast micrograph of free-floating and of tiny aggregated human breast adenocarcinoma cells (MCF7) cells (A) showing projections among cells (arrowhead) and ultrastructural (scanning electron microscopy, SEM) detail (B) of tunneling nanotubes (TNTs) protruding from a single cell. Scale bar: 4.5 μm. (C–E) SEM micrographs. The dimension of the aggregates is variable and the spatial organization of MCF7 cells is based on differing cell interactions. In smaller aggregate, few cells characterized by uniform dimension are in proximity (C) (white arrowhead), while in compacted ones, most of the cells are in close proximity or in contact (D) (white arrowhead), interacting (E) by short microvilli (mv) embedded in the dense amyloid matrix (white arrowhead). Scale bars: (C) 9 μm; (D) 9.5 μm; (E) 2 μm. (F,G) Transmission electron microscopy (TEM) analysis. MCF7 cell surfaces are characterized by microvilli and by TNT-like structures (arrowheads) reaching the neighboring cells. Scale bars: (F) 1.2 μm; (G) 2 μm. (H,I) TEM analysis. Cells are characterized by large dilated reticulum cisternae filled with spatially organized fibrillar material (H) (arrowheads) and released exosomes (I) (arrow). Scale bars: (H) 0.5 μm; (I) 0.6 μm. (J–O) Three day spheroid culture. (J,K) SEM analysis. MCF7 cells are assembled in small round spheroid uniform in size. Cells are in close proximity or in close contact and show different shape and size (arrowheads). Scale bars: (J) 15 μm; (K) 3.3 μm. (L–O) TEM analysis. Cells, polygonal in shape, are discontinuously connected (L–M), while they are distanced at the external side of the spheroid (N). These peripheral cells show numerous dilated rough endoplasmic reticulum (RER) cisternae filled with fibrillar material within the cytoplasm (N,O) (arrowheads). Scale bars: (L) 2.5 μm; (M) 4 μm; (N) 1.7 μm; (O) 0.6 μm.