Pathways Regulating Spheroid Formation of Human Follicular Thyroid Cancer Cells under Simulated Microgravity Conditions: A Genetic Approach.

Stefan Riwaldt,Thomas Corydon,Daniela Grimm,Manfred Infanger,Elisabeth Warnke,Anita Dittrich,Jessica Pietsch,Sascha Kopp,Johann Bauer,Markus Wehland,Nils Magnusson,Lasse Slumstrup

doi:10.3390/ijms17040528

Abstract

Microgravity induces three-dimensional (3D) growth in numerous cell types. Despite substantial efforts to clarify the underlying mechanisms for spheroid formation, the precise molecular pathways are still not known. The principal aim of this paper is to compare static 1g-control cells with spheroid forming (MCS) and spheroid non-forming (AD) thyroid cancer cells cultured in the same flask under simulated microgravity conditions. We investigated the morphology and gene expression patterns in human follicular thyroid cancer cells (UCLA RO82-W-1 cell line) after a 24 h-exposure on the Random Positioning Machine (RPM) and focused on 3D growth signaling processes. After 24 h, spheroid formation was observed in RPM-cultures together with alterations in the F-actin cytoskeleton. qPCR indicated more changes in gene expression in MCS than in AD cells. Of the 24 genes analyzed VEGFA, VEGFD, MSN, and MMP3 were upregulated in MCS compared to 1g-controls, whereas ACTB, ACTA2, KRT8, TUBB, EZR, RDX, PRKCA, CAV1, MMP9, PAI1, CTGF, MCP1 were downregulated. A pathway analysis revealed that the upregulated genes code for proteins, which promote 3D growth (angiogenesis) and prevent excessive accumulation of extracellular proteins, while genes coding for structural proteins are downregulated. Pathways regulating the strength/rigidity of cytoskeletal proteins, the amount of extracellular proteins, and 3D growth may be involved in MCS formation.

Highlights

Altered gravity conditions, such as real or simulated microgravity, offer new and unique approaches to cell biology in general and tissue engineering in particular
After a 24 h-exposure of UCLA RO82-W-1 follicular thyroid cancer cells to the random positioning machine (RPM), two types of growth were observed: irrespectively of the culture dish used, one part of the cells grew as 3D
Differences were considered significant at p < 0.05. This is the first study, using pathway analyses programs to investigate the molecular mechanisms responsible for 3D growth of follicular thyroid cancer cells grown under conditions of simulated microgravity for 24 h on the RPM

Summary

Introduction

Altered gravity conditions, such as real or simulated microgravity (μg), offer new and unique approaches to cell biology in general and tissue engineering in particular. We have exposed different healthy and malignant human thyroid cell lines to both real (on parabolic flight campaigns and on the SimBox/Shenzhou-8 Space Mission) and simulated (RPM) μg for different time periods [2,3,4,8,10,11,12,13,14]. These studies showed that healthy and malignant human thyroid cell lines share the capability to form 3D multicellular spheroids (MCS) after exposure to annulled gravity conditions for various time periods [10,11,12]. By investigating formation and growth of MCS, it might be possible to learn about the formation of metastases and to find new, so far neglected targets for cancer therapy/suppression

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