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

Sascha Kopp,Markus Wehland,Lasse Slumstrup,Ganna Aleshcheva,Jayashree Sahana,Thomas J Corydon,Johann Bauer,Nils E Magnusson,Daniela Grimm,Tawhidul Islam,Manfred Infanger

doi:10.1038/srep26887

Abstract

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.

Highlights

Many cell types form three-dimensional aggregates (MCS; multicellular spheroids), when they are cultured under microgravity
Phase contrast microscopy revealed epithelial-like MCF-7 cells growing in monolayers under normal static 1 g-conditions (Fig. 1A,C,E,G)
MCF-7 cells exposed to the Random Positioning Machine (RPM) for 2 h, 4 h, and 16 h showed no three-dimensional growth and only an adherent phenotype (Fig. 1B,D,F), whereas after a 24 h-RPM-exposure small compact round three-dimensional (3D) multicellular spheroids (MCS) were found floating in the supernatant (Fig. 1H)

Summary

Introduction

Many cell types form three-dimensional aggregates (MCS; multicellular spheroids), when they are cultured under microgravity. Known molecular targets (e.g. VEGF, VEGFR, HER2/neu) for approved drugs (e.g. tyrosine kinase inhibitors like sorafenib), or approved therapeutic antibodies (e.g. bevacizumab, ramucirumab, trastuzumab) are proteins, which are predominantly expressed in breast cancer cells and are simultaneously involved in promoting cell growth or apoptosis[3,4]. It is difficult at the current state of technology to apply the optimal cocktail of drugs to hit all cancer cells of any given patient. These morhological differences were accompanied by changes in biological processes such as proliferation and apoptosis as well as signaling pathways[18]

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