MCF7 human breast cancer cells were cultured under normal gravity (1 g) and on a random positioning machine (RPM) preventing sedimentation. After 2 weeks, adherent 1 g-control and adherent RPM cells (AD) as well as multicellular spheroids (MCS) were harvested. AD and MCS had been exposed to the RPM in the same culture flask. In a subsequent proteome analysis, the majority of the proteins detected showed similar label-free quantification (LFQ) scores in each of the respective subpopulations, but in both AD or MCS cultures, proteins were also found whose LFQs deviated at least twofold from their counterparts in the 1 g-control cells. They included the cell junction protein E-cadherin, which was diminished in MCS cells, where proteins of the E-cadherin autodegradation pathway were enhanced and c-Src (proto-oncogene tyrosine-protein kinase c-Src) was detected. Spheroid formation was prevented by inhibition of c-Src but promoted by antibodies blocking E-cadherin activity. An interaction analysis of the detected proteins that are involved in forming and regulating junctions or adhesion complexes and in E-cadherin autodegradation indicated connections between the two protein groups. This suggests that the balance of proteins that up- or downregulate E-cadherin mediates the tendency of MCF7 cells to form MCS during RPM exposure.

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.

Prostate cancer is one of the leading causes of cancer mortality in men worldwide. An unusual but unique environment for studying tumor cell processes is provided by microgravity, either in space or simulated by ground-based devices like a random positioning machine (RPM). In this study, prostate adenocarcinoma-derived PC-3 cells were cultivated on an RPM for time periods of 3 and 5 days. We investigated the genes associated with the cytoskeleton, focal adhesions, extracellular matrix, growth, survival, angiogenesis, and metastasis. The gene expression of signaling factors of the vascular endothelial growth factor (VEGF), mitogen-activated protein kinase (MAPK), and PI3K/AKT/mTOR (PAM) pathways was investigated using qPCR. We performed immunofluorescence to study the cytoskeleton, histological staining to examine the morphology, and a time-resolved immunofluorometric assay to analyze the cell culture supernatants. When PC-3 cells were exposed to simulated microgravity (s-µg), some cells remained growing as adherent cells (AD), while most cells detached from the cell culture flask bottom and formed multicellular spheroids (MCS). After 3-day RPM exposure, PC-3 cells revealed significant downregulation of the VEGF, SRC1, AKT, MTOR, and COL1A1 gene expression in MCS, whereas FLT1, RAF1, MEK1, ERK1, FAK1, RICTOR, ACTB, TUBB, and TLN1 mRNAs were not significantly changed. ERK2 and TLN1 were elevated in AD, and FLK1, LAMA3, COL4A5, FN1, VCL, CDH1, and NGAL mRNAs were significantly upregulated in AD and MCS after 3 days. After a 5-day culture in s-µg, the PC-3 cells showed significant downregulations of VEGF mRNA in AD and MCS, and FN1, CDH1, and LAMA3 in AD and SCR1 in MCS. In addition, we measured significant upregulations in FLT1, AKT, ERK1, ERK2, LCN2, COL1A1, TUBB, and VCL mRNAs in AD and MCS, and increases in FLK1, FN1, and COL4A5 in MCS as well as LAMB2, CDH1, RAF1, MEK1, SRC1, and MTOR mRNAs in AD. FAK1 and RICTOR were not altered by s-µg. In parallel, the secretion rate of VEGFA and NGAL proteins decreased. Cytoskeletal alterations (F-actin) were visible, as well as a deposition of collagen in the MCS. In conclusion, RPM-exposure of PC-3 cells induced changes in their morphology, cytoskeleton, and extracellular matrix protein synthesis, as well as in their focal adhesion complex and growth behavior. The significant upregulation of genes belonging to the PAM pathway indicated their involvement in the cellular changes occurring in microgravity.

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.

With the commercialization of spaceflight and the exploration of space, it is important to understand the changes occurring in human cells exposed to real microgravity (r-µg) conditions. We examined the influence of r-µg, simulated microgravity (s-µg, incubator random positioning machine (iRPM)), hypergravity (hyper-g), and vibration (VIB) on triple-negative breast cancer (TNBC) cells (MDA-MB-231 cell line) with the aim to study early changes in the gene expression of factors associated with cell adhesion, apoptosis, nuclear factor “kappa-light-chain-enhancer” of activated B-cells (NF-κB) and mitogen-activated protein kinase (MAPK) signaling. We had the opportunity to attend a parabolic flight (PF) mission and to study changes in RNA transcription in the MDA-MB cells exposed to PF maneuvers (29th Deutsches Zentrum für Luft- und Raumfahrt (DLR) PF campaign). PF maneuvers induced an early up-regulation of ICAM1, CD44 and ERK1 mRNAs after the first parabola (P1) and a delayed upregulation of NFKB1, NFKBIA, NFKBIB, and FAK1 after the last parabola (P31). ICAM-1, VCAM-1 and CD44 protein levels were elevated, whereas the NF-κB subunit p-65 and annexin-A2 protein levels were reduced after the 31st parabola (P31). The PRKCA, RAF1, BAX mRNA were not changed and cleaved caspase-3 was not detectable in MDA-MB-231 cells exposed to PF maneuvers. Hyper-g-exposure of the cells elevated the expression of CD44 and NFKBIA mRNAs, iRPM-exposure downregulated ANXA2 and BAX, whereas VIB did not affect the TNBC cells. The early changes in ICAM-1 and VCAM-1 and the rapid decrease in the NF-κB subunit p-65 might be considered as fast-reacting, gravity-regulated and cell-protective mechanisms of TNBC cells exposed to altered gravity conditions. This data suggest a key role for the detected gravity-signaling elements in three-dimensional growth and metastasis.