Microgravity induces changes in the cytoskeleton. This might have an impact on cells and organs of humans in space. Unfortunately, studies of cytoskeletal changes in microgravity reported so far are obligatorily based on the analysis of fixed cells exposed to microgravity during a parabolic flight campaign (PFC). This study focuses on the development of a compact fluorescence microscope (FLUMIAS) for fast live-cell imaging under real microgravity. It demonstrates the application of the instrument for on-board analysis of cytoskeletal changes in FTC-133 cancer cells expressing the Lifeact-GFP marker protein for the visualization of F-actin during the 24th DLR PFC and TEXUS 52 rocket mission. Although vibration is an inevitable part of parabolic flight maneuvers, we successfully for the first time report life-cell cytoskeleton imaging during microgravity, and gene expression analysis after the 31st parabola showing a clear up-regulation of cytoskeletal genes. Notably, during the rocket flight the FLUMIAS microscope reveals significant alterations of the cytoskeleton related to microgravity. Our findings clearly demonstrate the applicability of the FLUMIAS microscope for life-cell imaging during microgravity, rendering it an important technological advance in live-cell imaging when dissecting protein localization.

With the increasing number of spaceflights, it is crucial to understand the changes occurring in human cells exposed to real microgravity (r-µg) conditions. We tested the effect of r-µg on MCF-7 breast cancer cells with the objective to investigate cytoskeletal alterations and early changes in the gene expression of factors belonging to the cytoskeleton, extracellular matrix, focal adhesion, and cytokines. In the Technische Experimente unter Schwerelosigkeit (TEXUS) 54 rocket mission, we had the opportunity to conduct our experiment during 6 min of r-µg and focused on cytoskeletal alterations of MCF-7 breast cancer cells expressing the Lifeact-GFP marker protein for the visualization of F-actin as well as the mCherry-tubulin fusion protein using the Fluorescence Microscopy Analysis System (FLUMIAS) for fast live-cell imaging under r-µg. Moreover, in a second mission we investigated changes in RNA transcription and morphology in breast cancer cells exposed to parabolic flight (PF) maneuvers (31st Deutsches Zentrum für Luft- und Raumfahrt (DLR) PF campaign). The MCF-7 cells showed a rearrangement of the F-actin and tubulin with holes, accumulations in the tubulin network, and the appearance of filopodia- and lamellipodia-like structures in the F-actin cytoskeleton shortly after the beginning of the r-µg period. PF maneuvers induced an early up-regulation of KRT8, RDX, TIMP1, CXCL8 mRNAs, and a down-regulation of VCL after the first parabola. E-cadherin protein was significantly reduced and is involved in cell adhesion processes, and plays a significant role in tumorigenesis. Changes in the E-cadherin protein synthesis can lead to tumor progression. Pathway analyses indicate that VCL protein has an activating effect on CDH1. In conclusion, live-cell imaging visualized similar changes as those occurring in thyroid cancer cells in r-µg. This result indicates the presence of a common mechanism of gravity perception and sensation.

Human follicular thyroid cancer cells (FTC-133) were sent to space via a sounding rocket during the TEXUS-53 mission to determine the impact of short-term microgravity on these cells. To enable cell culture and fixation in real microgravity, an automated experiment container (EC) was constructed. In order to ensure safe cell culture, cell-chambers consisting of polycarbonate (PC) material were used. They were highly biocompatible as proved by measuring cell survival using Annexin V flow cytometry. In the follow-up experiment, FTC-133 cells were sent to space via a sounding rocket and were fixed before and after the microgravity (µg) phase with RNAlater. In addition, cells were tested for reactions on hypergravity (hyper-g) as much as 18 g to determine whether worst case acceleration during launch can have an influence on the cells. We investigated genes belonging to biological processes such as cytoskeleton, cell adhesion, tumor growth, angiogenesis and apoptosis. Pathway analyses revealed central functions of VEGFA and EGF. EGF upregulates aspartate beta-hydroxylase (ASPH) which is influencing CASP3. Hyper-g induced a significant up-regulation of TUBB1, VIM, RDX, CAV1, VEGFA and BCL2. FTC-133 cells grown in an automated EC exposed to µg revealed moderate gene expression changes indicating their survival in orbit.

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