Establishment of a Human Immunocompetent 3D Tissue Model to Enable the Long-Term Examination of Biofilm-Tissue Interactions.

Different studies suggest an impact of biofilms on carcinogenic lesion formation in varying human tissues. However, the mechanisms of cancer formation are difficult to examine in vivo as well as in vitro. Cell culture approaches, in most cases, are unable to keep a bacterial steady state without any overgrowth. In our approach, we aimed to develop an immunocompetent 3D tissue model which can mitigate bacterial outgrowth. We established a three-dimensional (3D) co-culture of human primary fibroblasts with pre-differentiated THP-1-derived macrophages on an SIS-muc scaffold which was derived by decellularisation of a porcine intestine. After establishment, we exposed the tissue models to define the biofilms of the Pseudomonas spec. and Staphylococcus spec. cultivated on implant mesh material. After 3 days of incubation, the cell culture medium in models with M0 and M2 pre-differentiated macrophages presented a noticeable turbidity, while models with M1 macrophages presented no noticeable bacterial growth. These results were validated by optical density measurements and a streak test. Immunohistology and immunofluorescent staining of the tissue presented a positive impact of the M1 macrophages on the structural integrity of the tissue model. Furthermore, multiplex ELISA highlighted the increased release of inflammatory cytokines for all the three model types, suggesting the immunocompetence of the developed model. Overall, in this proof-of-principle study, we were able to mitigate bacterial overgrowth and prepared a first step for the development of more complex 3D tissue models to understand the impact of biofilms on carcinogenic lesion formation.

Dexamethasone Selectively Inhibits Detachment of Metastatic Thyroid Cancer Cells during Random Positioning.

We recently reported that synthetic glucocorticoid dexamethasone (DEX) is able to suppress metastasis-like spheroid formation in a culture of follicular thyroid cancer (FTC)-133 cells cultured under random positioning. We now show that this inhibition was selective for two metastatic thyroid carcinoma cells, FTC-133 and WRO, whereas benign Nthy-ori 3-1 thyrocytes and recurrent ML-1 follicular thyroid cancer cells were not affected by DEX. We then compare Nthy-ori 3-1 and FTC-133 cells concerning their adhesion and mechanosignaling. We demonstrate that DEX disrupts random positioning-triggered p38 stress signaling in FTC-133 cells, thereby antagonizing a variety of biological functions. Thus, DEX treatment of FTC-133 cells is associated with increased adhesiveness, which is mainly caused by the restored, pronounced formation of a normal number of tight junctions. Moreover, we show that Nthy-ori 3-1 and ML-1 cells upregulate the anti-adhesion protein mucin-1 during random positioning, presumably as a protection against mechanical stress. In summary, mechanical stress seems to be an important component in this metastasis model system that is processed differently by metastatic and healthy cells. The balance between adhesion, anti-adhesion and cell-cell connections enables detachment of adherent human cells on the random positioning machine-or not, allowing selective inhibition of thyroid in vitro metastasis by DEX.

Tumor Models and Drug Targeting In Vitro-Where Are We Today? Where Do We Go from Here?

Cancer is one of the leading causes of death worldwide [...].

Structural and Molecular Changes of Human Chondrocytes Exposed to the Rotating Wall Vessel Bioreactor.

Over the last 30 years, the prevalence of osteoarthritis (OA), a disease characterized by a loss of articular cartilage, has more than doubled worldwide. Patients suffer from pain and progressive loss of joint function. Cartilage is an avascular tissue mostly consisting of extracellular matrix with embedded chondrocytes. As such, it does not regenerate naturally, which makes an early onset of OA prevention and treatment a necessity to sustain the patients' quality of life. In recent years, tissue engineering strategies for the regeneration of cartilage lesions have gained more and more momentum. In this study, we aimed to investigate the scaffold-free 3D cartilage tissue formation under simulated microgravity in the NASA-developed rotating wall vessel (RWV) bioreactor. For this purpose, we cultured both primary human chondrocytes as well as cells from the immortalized line C28/I2 for up to 14 days on the RWV and analyzed tissue morphology, development of apoptosis, and expression of cartilage-specific proteins and genes by histological staining, TUNEL-assays, immunohistochemical detection of collagen species, and quantitative real-time PCR, respectively. We observed spheroid formation in both cell types starting on day 3. After 14 days, constructs from C28/I2 cells had diameters of up to 5 mm, while primary chondrocyte spheroids were slightly smaller with 3 mm. Further inspection of the 14-day-old C28/I2 spheroids revealed a characteristic cartilage morphology with collagen-type 1, -type 2, and -type 10 positivity. Interestingly, these tissues were less susceptible to RWV-induced differential gene expression than those formed from primary chondrocytes, which showed significant changes in the regulation of IL6, ACTB, TUBB, VIM, COL1A1, COL10A1, MMP1, MMP3, MMP13, ITGB1, LAMA1, RUNX3, SOX9, and CASP3 gene expression. These diverging findings might reflect the differences between primary and immortalized cells. Taken together, this study shows that simulated microgravity using the RWV bioreactor is suitable to engineer dense 3D cartilage-like tissue without addition of scaffolds or any other artificial materials. Both primary articular cells and the stable chondrocyte cell line C28/I2 formed 3D neocartilage when exposed for 14 days to an RWV.

Three-Dimensional Growth of Prostate Cancer Cells Exposed to Simulated Microgravity.

Prostate cancer metastasis has an enormous impact on the mortality of cancer patients. Factors involved in cancer progression and metastasis are known to be key players in microgravity (µg)-driven three-dimensional (3D) cancer spheroid formation. We investigated PC-3 prostate cancer cells for 30 min, 2, 4 and 24 h on the random positioning machine (RPM), a device simulating µg on Earth. After a 24 h RPM-exposure, the cells could be divided into two groups: one grew as 3D multicellular spheroids (MCS), the other one as adherent monolayer (AD). No signs of apoptosis were visible. Among others, we focused on cytokines involved in the events of metastasis and MCS formation. After 24 h of exposure, in the MCS group we measured an increase in ACTB, MSN, COL1A1, LAMA3, FN1, TIMP1, FLT1, EGFR1, IL1A, IL6, CXCL8, and HIF1A mRNA expression, and in the AD group an elevation of LAMA3, COL1A1, FN1, MMP9, VEGFA, IL6, and CXCL8 mRNAs compared to samples subjected to 1 g conditions. Significant downregulations in AD cells were detected in the mRNA levels of TUBB, KRT8, IL1B, IL7, PIK3CB, AKT1 and MTOR after 24 h. The release of collagen-1α1 and fibronectin protein in the supernatant was decreased, whereas the secretion of IL-6 was elevated in 24 h RPM samples. The secretion of IL-1α, IL-1ß, IL-7, IL-2, IL-8, IL-17, TNF-α, laminin, MMP-2, TIMP-1, osteopontin and EGF was not significantly altered after 24 h compared to 1 g conditions. The release of soluble factors was significantly reduced after 2 h (IL-1α, IL-2, IL-7, IL-8, IL-17, TNF-α, collagen-1α1, MMP-2, osteopontin) and elevated after 4 h (IL-1ß, IL-2, IL-6, IL-7, IL-8, TNF-α, laminin) in RPM samples. Taken together, simulated µg induced 3D growth of PC-3 cancer cells combined with a differential expression of the cytokines IL-1α, IL-1ß, IL-6 and IL-8, supporting their involvement in growth and progression of prostate cancer cells.

The CellBox-2 Mission to the International Space Station: Thyroid Cancer Cells in Space.

A spaceflight to the International Space Station (ISS) is a dream of many researchers. We had the chance to investigate the effect of real microgravity (CellBox-2 Space mission) on the transcriptome and proteome of FTC-133 human follicular thyroid cancer cells (TCC). The cells had been sent to the ISS by a Falcon 9 rocket of SpaceX CRS-13 from Cape Canaveral (United States) and cultured in six automated hardware units on the ISS before they were fixed and returned to Earth. Multicellular spheroids (MCS) were detectable in all spaceflight hardware units. The VCL, PXN, ITGB1, RELA, ERK1 and ERK2 mRNA levels were significantly downregulated after 5 days in space in adherently growing cells (AD) and MCS compared with ground controls (1g), whereas the MIK67 and SRC mRNA levels were both suppressed in MCS. By contrast, the ICAM1, COL1A1 and IL6 mRNA levels were significantly upregulated in AD cells compared with 1g and MCS. The protein secretion measured by multianalyte profiling technology and enzyme-linked immunosorbent assay (AngiogenesisMAP®, extracellular matrix proteins) was not significantly altered, with the exception of elevated angiopoietin 2. TCC in space formed MCS, and the response to microgravity was mainly anti-proliferative. We identified ERK/RELA as a major microgravity regulatory pathway.

Role of Apoptosis in Wound Healing and Apoptosis Alterations in Microgravity.

Functioning as the outermost self-renewing protective layer of the human organism, skin protects against a multitude of harmful biological and physical stimuli. Consisting of ectodermal, mesenchymal, and neural crest-derived cell lineages, tissue homeostasis, and signal transduction are finely tuned through the interplay of various pathways. A health problem of astronauts in space is skin deterioration. Until today, wound healing has not been considered as a severe health concern for crew members. This can change with deep space exploration missions and commercial spaceflights together with space tourism. Albeit the molecular process of wound healing is not fully elucidated yet, there have been established significant conceptual gains and new scientific methods. Apoptosis, e.g., programmed cell death, enables orchestrated development and cell removal in wounded or infected tissue. Experimental designs utilizing microgravity allow new insights into the role of apoptosis in wound healing. Furthermore, impaired wound healing in unloading conditions would depict a significant challenge in human-crewed exploration space missions. In this review, we provide an overview of alterations in the behavior of cutaneous cell lineages under microgravity in regard to the impact of apoptosis in wound healing. We discuss the current knowledge about wound healing in space and simulated microgravity with respect to apoptosis and available therapeutic strategies.

Alterations of Growth and Focal Adhesion Molecules in Human Breast Cancer Cells Exposed to the Random Positioning Machine.

In this study, we evaluated changes in focal adhesions (FAs) in two types of breast cancer cell (BCC) lines (differentiated MCF-7 and the triple-negative MDA-MB-231 cell line) exposed to simulated microgravity (s-µg) created by a random positioning machine (RPM) for 24 h. After exposure, the BCC changed their growth behavior and exhibited two phenotypes in RPM samples: one portion of the cells grew as a normal two-dimensional monolayer [adherent (AD) BCC], while the other portion formed three-dimensional (3D) multicellular spheroids (MCS). After 1 h and 30 min (MDA-MB-231) and 1 h 40 min (MCF-7), the MCS adhered completely to the slide flask bottom. After 2 h, MDA-MB-231 MCS cells started to migrate, and after 6 h, a large number of the cells had left the MCS and continued to grow in a scattered pattern, whereas MCF-7 cells were growing as a confluent monolayer after 6 h and 24 h. We investigated the genes associated with the cytoskeleton, the extracellular matrix and FAs. ACTB, TUBB, FN1, FAK1, and PXN gene expression patterns were not significantly changed in MDA-MB-231 cells, but we observed a down-regulation of LAMA3, ITGB1 mRNAs in AD cells and of ITGB1, TLN1 and VCL mRNAs in MDA-MB-231 MCS. RPM-exposed MCF-7 cells revealed a down-regulation in the gene expression of FAK1, PXN, TLN1, VCL and CDH1 in AD cells and PXN, TLN and CDH1 in MCS. An interaction analysis of the examined genes involved in 3D growth and adhesion indicated a central role of fibronectin, vinculin, and E-cadherin. Live cell imaging of eGFP-vinculin in MCF-7 cells confirmed these findings. ß-catenin-transfected MCF-7 cells revealed a nuclear expression in 1g and RPM-AD cells. The target genes BCL9, MYC and JUN of the Wnt/ß-catenin signaling pathway were differentially expressed in RPM-exposed MCF-7 cells. These findings suggest that vinculin and ß-catenin are key mediators of BCC to form MCS during 24 h of RPM-exposure.

SARS-CoV-2 and hypertension.

The objective of this review is to give an overview of the pathophysiological effects of the Coronavirus Disease 2019 (COVID-19) in relation to hypertension (HT), with a focus on the Renin-Angiotensin-Aldosterone System (RAAS) and the MAS receptor. HT is a multifactorial disease and a public health burden, as it is a risk factor for diseases like stroke, coronary artery disease, and heart failure, leading to 10.4 million deaths yearly. Blood pressure is regulated by the RAAS. The system consists of two counter-regulatory axes: ACE/ANG-II/AT1 R and ACE2/ANG-(1-7)/MAS. The main regulatory protein in balancing the RAAS is angiotensin-converting enzyme 2 (ACE2). The protein also functions as the main mediator of endocytosis of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into the host cell. SARS-CoV-2 is the cause of COVID-19 and has caused a worldwide pandemic; however, the treatment and prophylaxis of COVID-19 are limited. Several drugs and vaccines are currently being tested in clinical trials with a few already approved by EMA and FDA. HT is a major risk factor regarding the severity and fatality of COVID-19, and the RAAS plays an important role in COVID-19 infection since SARS-CoV-2 can lead to a dysregulation of the system by reducing the ACE2 expression. The exact mechanisms of HT in relation to COVID-19 remain uncertain, and more research is needed for further elucidation.

Tissue Engineering of Cartilage Using a Random Positioning Machine.

Articular cartilage is a skeletal tissue of avascular nature and limited self-repair capacity. Cartilage-degenerative diseases, such as osteoarthritis (OA), are difficult to treat and often necessitate joint replacement surgery. Cartilage is a tough but flexible material and relatively easy to damage. It is, therefore, of high interest to develop methods allowing chondrocytes to recolonize, to rebuild the cartilage and to restore joint functionality. Here we studied the in vitro production of cartilage-like tissue using human articular chondrocytes exposed to the Random Positioning Machine (RPM), a device to simulate certain aspects of microgravity on Earth. To screen early adoption reactions of chondrocytes exposed to the RPM, we performed quantitative real-time PCR analyses after 24 h on chondrocytes cultured in DMEM/F-12. A significant up-regulation in the gene expression of IL6, RUNX2, RUNX3, SPP1, SOX6, SOX9, and MMP13 was detected, while the levels of IL8, ACAN, PRG4, ITGB1, TGFB1, COL1A1, COL2A1, COL10A1, SOD3, SOX5, MMP1, and MMP2 mRNAs remained unchanged. The STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis demonstrated among others the importance of these differentially regulated genes for cartilage formation. Chondrocytes grown in DMEM/F-12 medium produced three-dimensional (3D) spheroids after five days without the addition of scaffolds. On day 28, the produced tissue constructs reached up to 2 mm in diameter. Using specific chondrocyte growth medium, similar results were achieved within 14 days. Spheroids from both types of culture media showed the typical cartilage morphology with aggrecan positivity. Intermediate filaments form clusters under RPM conditions as detected by vimentin staining after 7 d and 14 d. Larger meshes appear in the network in 28-day samples. Furthermore, they were able to form a confluent chondrocyte monolayer after being transferred back into cell culture flasks in 1 g conditions showing their suitability for transplantation into joints. Our results demonstrate that the cultivation medium has a direct influence on the velocity of tissue formation and tissue composition. The spheroids show properties that make them interesting candidates for cellular cartilage regeneration approaches in trauma and OA therapy.

Breast Cancer Cells in Microgravity: New Aspects for Cancer Research.

Breast cancer is the leading cause of cancer death in females. The incidence has risen dramatically during recent decades. Dismissed as an "unsolved problem of the last century", breast cancer still represents a health burden with no effective solution identified so far. Microgravity (µg) research might be an unusual method to combat the disease, but cancer biologists decided to harness the power of µg as an exceptional method to increase efficacy and precision of future breast cancer therapies. Numerous studies have indicated that µg has a great impact on cancer cells; by influencing proliferation, survival, and migration, it shifts breast cancer cells toward a less aggressive phenotype. In addition, through the de novo generation of tumor spheroids, µg research provides a reliable in vitro 3D tumor model for preclinical cancer drug development and to study various processes of cancer progression. In summary, µg has become an important tool in understanding and influencing breast cancer biology.

Science between Bioreactors and Space Research-Response to Comments by Joseph J. Bevelacqua et al. on "Dexamethasone Inhibits Spheroid Formation of Thyroid Cancer Cells Exposed to Simulated Microgravity".

We would like to thank Bevelacqua et al [...].

The effects of microgravity on differentiation and cell growth in stem cells and cancer stem cells.

A spaceflight has enormous influence on the health of space voyagers due to the combined effects of microgravity and cosmic radiation. Known effects of microgravity (µg) on cells are changes in differentiation and growth. Considering the commercialization of spaceflight, future space exploration, and long-term manned flights, research focusing on differentiation and growth of stem cells and cancer cells exposed to real (r-) and simulated (s-) µg is of high interest for regenerative medicine and cancer research. In this review, we focus on platforms to study r- and s-µg as well as the impact of µg on cancer stem cells in the field of gastrointestinal cancer, lung cancer, and osteosarcoma. Moreover, we review the current knowledge of different types of stem cells exposed to µg conditions with regard to differentiation and engineering of cartilage, bone, vasculature, heart, skin, and liver constructs.

Dexamethasone Inhibits Spheroid Formation of Thyroid Cancer Cells Exposed to Simulated Microgravity.

Detachment and the formation of spheroids under microgravity conditions can be observed with various types of intrinsically adherent human cells. In particular, for cancer cells this process mimics metastasis and may provide insights into cancer biology and progression that can be used to identify new drug/target combinations for future therapies. By using the synthetic glucocorticoid dexamethasone (DEX), we were able to suppress spheroid formation in a culture of follicular thyroid cancer (FTC)-133 cells that were exposed to altered gravity conditions on a random positioning machine. DEX inhibited the growth of three-dimensional cell aggregates in a dose-dependent manner. In the first approach, we analyzed the expression of several factors that are known to be involved in key processes of cancer progression such as autocrine signaling, proliferation, epithelial-mesenchymal transition, and anoikis. Wnt/ß-catenin signaling and expression patterns of important genes in cancer cell growth and survival, which were further suggested to play a role in three-dimensional aggregation, such as NFKB2, VEGFA, CTGF, CAV1, BCL2(L1), or SNAI1, were clearly affected by DEX. Our data suggest the presence of a more complex regulation network of tumor spheroid formation involving additional signal pathways or individual key players that are also influenced by DEX.

Simulated Microgravity Influences VEGF, MAPK, and PAM Signaling in Prostate Cancer Cells.

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.

Short-Term Microgravity Influences Cell Adhesion in Human Breast Cancer Cells.

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.

Beneficial Effects of Low Frequency Vibration on Human Chondrocytes in Vitro.

BACKGROUND/AIMS: In articular cartilage, chondrocytes are the predominant cell type. A long-term stay in space can lead to bone loss and cartilage breakdown. Due to the poor regenerative capacity of cartilage, this may impair the crewmembers' mobility and influence mission activities. Beside microgravity other factors such as cosmic radiation and vibration might be important for cartilage degeneration. Vibration at different frequencies showed various effects on cartilage in vivo, but knowledge about its impact on chondrocytes in vitro is sparse. METHODS: Human chondrocytes were exposed to a vibration device, simulating the vibration profile occurring during parabolic flights, for 24 h (VIB) and compared to static controls. Phase-contrast microscopy, immunofluorescence, F-actin and TUNEL staining as well as quantitative real-time PCR were performed to examine effects on morphology, cell viability and shape as well as gene expression. The results were compared to earlier studies using semantic analyses. RESULTS: No morphological changes or cytoskeletal alterations were observed in VIB and no apoptotic cells were found. A reorganization and increase in fibronectin were detected in VIB samples by immunofluorescence technique. PXN, VCL, ANXA1, ANXA2, BAX, and BCL2 revealed differential regulations. CONCLUSION: Long-term VIB did not damage human chondrocytes in vitro. The reduction of ANXA2, and up-regulation of ANXA1, PXN and VCL mRNAs suggest that long-term vibration might even positively influence cultured chondrocytes.

Real Microgravity Influences the Cytoskeleton and Focal Adhesions in Human Breast Cancer Cells.

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.

Fighting Thyroid Cancer with Microgravity Research.

Microgravity in space or simulated by special ground-based devices provides an unusual but unique environment to study and influence tumour cell processes. By investigating thyroid cancer cells in microgravity for nearly 20 years, researchers got insights into tumour biology that had not been possible under normal laboratory conditions: adherently growing cancer cells detach from their surface and form three-dimensional structures. The cells included in these multicellular spheroids (MCS) were not only altered but behave also differently to those grown in flat sheets in normal gravity, more closely mimicking the conditions in the human body. Therefore, MCS became an invaluable model for studying metastasis and developing new cancer treatment strategies via drug targeting. Microgravity intervenes deeply in processes such as apoptosis and in structural changes involving the cytoskeleton and the extracellular matrix, which influence cell growth. Most interestingly, follicular thyroid cancer cells grown under microgravity conditions were shifted towards a less-malignant phenotype. Results from microgravity research can be used to rethink conventional cancer research and may help to pinpoint the cellular changes that cause cancer. This in turn could lead to novel therapies that will enhance the quality of life for patients or potentially develop new preventive countermeasures.

Growth of Endothelial Cells in Space and in Simulated Microgravity - a Comparison on the Secretory Level.

BACKGROUND/AIMS: Endothelial cells exposed to the Random Positioning Machine (RPM) reveal three different phenotypes. They grow as a two-dimensional monolayer and form three-dimensional (3D) structures such as spheroids and tubular constructs. As part of the ESA-SPHEROIDS project we want to understand how endothelial cells (ECs) react and adapt to long-term microgravity. METHODS: During a spaceflight to the International Space Station (ISS) and a subsequent stay onboard, human ECs (EA.hy926 cell line) were cultured for 12 days in real microgravity inside an automatic flight hardware, specially designed for use in space. ECs were cultivated in the absence or presence of vascular endothelial growth factor, which had demonstrated a cell-protective effect on ECs exposed to an RPM simulating microgravity. After cell fixation in space and return of the samples, we examined cell morphology and analyzed supernatants by Multianalyte Profiling technology. RESULTS: The fixed samples comprised 3D multicellular spheroids and tube-like structures in addition to monolayer cells, which are exclusively observed during growth under Earth gravity (1g). Within the 3D aggregates we detected enhanced collagen and laminin. The supernatant analysis unveiled alterations in secretion of several growth factors, cytokines, and extracellular matrix components as compared to cells cultivated at 1g or on the RPM. This confirmed an influence of gravity on interacting key proteins and genes and demonstrated a flight hardware impact on the endothelial secretome. CONCLUSION: Since formation of tube-like aggregates was observed only on the RPM and during spaceflight, we conclude that microgravity may be the major cause for ECs' 3D aggregation.