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Many cell types form three-dimensional aggregates (MCS; multicellular spheroids), when they are cultured under microgravity. MCS often resemble the organ, from which the cells have been derived. In this study we investigated human MCF-7 breast cancer cells after a 2 h-, 4 h-, 16 h-, 24 h- and 5d-exposure to a Random Positioning Machine (RPM) simulating microgravity. At 24 h few small compact MCS were detectable, whereas after 5d many MCS were floating in the supernatant above the cells, remaining adherently (AD). The MCS resembled the ducts formed  in vivo  by human epithelial breast cells. In order to clarify the underlying mechanisms, we harvested MCS and AD cells separately from each RPM-culture and measured the expression of 29 selected genes with a known involvement in MCS formation. qPCR analyses indicated that cytoskeletal genes were unaltered in short-term samples.  IL8, VEGFA , and  FLT1  were upregulated in 2 h/4 h AD-cultures. The  ACTB, TUBB, EZR, RDX, FN1, VEGFA, FLK1 Casp9, Casp3, PRKCA  mRNAs were downregulated in 5d-MCS-samples.  ESR1  was upregulated in AD, and  PGR1  in both phenotypes after 5d. A pathway analysis revealed that the corresponding gene products are involved in organization and regulation of the cell shape, in cell tip formation and membrane to membrane docking.

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Identifications of novel mechanisms in breast cancer cells involving duct-like multicellular spheroid formation after exposure to the Random Positioning Machine