Microgravity-based Modulation of VEGF Expression in Human Thyroid Carcinoma Cells

• ¹ Medizinische Fakultät, Universitätsklinikum Magdeburg, Germany
• ² Max-Planck-Institut für Biochemie, Germany
• ³ Aarhus University, Denmark

ABSTRACT When human thyroid carcinoma cells are exposed to microgravity some adherent cells detach from the bottom of the culture flask and grow into floating multicellular spheroids. It is assumed that this process mimics metastasis. The spheroids generated are more complex than a cell monolayer and exhibits properties of small tumours, which cannot be found in a normal cell culture. Therefore, tumour cell spheroids are important structures in cancer research, for example to find new targets for anticancer therapy. We investigated the effect of real microgravity on the transcriptome and proteome of FTC-133 human follicular thyroid cancer cells returned from the CellBox-2 Space mission. The cells had been cultured for 5 and 10 days in six automated hardware units on the International Space Station before they were fixed and sent back to Earth. Spheroids were formed in all of the hardware units. They differed from spheroids generated under simulated microgravity on a Random Positioning Machine (RPM) mainly by an enhanced release of the vascular endothelial growth factor (VEGF). VEGF has a key role in the production of tumour vasculature. Its enhanced production may hint at a different angiogenic potential of thyroid cancer cells in Space. INTRODUCTION Tumour growth and development depend on neovascularization, that is strictly regulated by growth factors binding to receptor tyrosine kinases. Vascular endothelial growth factor (VEGF) is essential for the formation of new vessels in different developmental processes, pathological conditions, tumour growth, and metastasis (Lin and Chao, 2005). VEGF is up-regulated in thyroid malignancies (Karaca et al., 2011) and initiates an endothelial cell-specific signalling pathway via Flk-1/KDR which is required for blood vessel formation. Inhibition of this pathway has become an important therapeutic target as it blocks pathological angiogenesis in growing tumours, leading to stasis or regression of tumour growth (McMahon, 2000). Exposure to prolonged microgravity (µg) can influence fundamental processes in human cells including cancer cells. Numerous studies that were performed in the simulated µg (s-µg) of a Random Positioning Machine (RPM) or clinostat have revealed alterations of apoptosis, proliferation, differentiation, growth behaviour, cell adhesion, extracellular matrix, and cytoskeleton (Grimm et al., 2002; Warnke et al., 2014). Moreover, the scaffold-free formation of multicellular spheroids (MCS) was observed for many times (Pietsch et al., 2010; Pietsch et al., 2011; Grosse et al., 2012; Pietsch et al., 2012; Pietsch et al., 2013; Bauer et al., 2017; Sahana et al., 2018). In 2017, the CellBox-2 experiment was set up to investigate FTC-133 cells in real µg (r-µg) in Space during a long-term-experiment of 5 and 10 days. In the current study, we analysed the VEGF gene expression and its release. To evaluate differences of r-µg and s-µg, we performed an additional experiment with an RPM using the protocol and timeline of the CellBox-2 mission. MATERIALS AND METHODS Cell culture The human follicular thyroid cancer cell line FTC-133 were cultured in RPMI-1640 medium supplemented with 10% FCS and 1% penicillin/streptomycin at 37°C and 5% CO2 until use for the experiments. Flight preparation and CellBox-2 mission Cells were sent to the International Space Station (ISS) by a Falcon 9 rocket of SpaceX CRS-13 from Cape Canaveral (USA). 2d before launch, the FTC-133 cells were filled in 6 automated hardware units (FM 1-6, Fig. 1) with a cell suspension containing 1×106 cells and were kept at 23°C. Each hardware unit consists of a cell cultivation chamber, two reserve tanks for medium and fixative and a pump for fluid exchange. 24h before launch, FM 1-6 were loaded into the rocket. 2d after launch, FM 1-6 were installed on the ISS marking the starting point of our experiment. The FTC-133 cells were stored at 23°C. After 5d in µg, the first medium exchange was performed automatically in all six FMs. Afterwards, the cells of FM 1-3 were fixed automatically with RNAlater. After another 5d, the procedure was repeated with FM 4-6. Finally, all FMs were stored at 4°C until their flight back to Earth. To investigate the effect of µg the same timeline and handling were used for the 1g-control group on ground. RPM Experiment The RPM experiment was performed as described in (Ma et al., 2014), but with the time and temperature profiles of the Space Experiment. RNA extraction and quantitative polymerase chain reaction (qPCR) RNA extraction and qPCR were performed as recently described in (Kopp et al., 2018). The sequences of the VEGFA forward and reverse primers were: 5`-GCGCTGATAGACATCCATGAAC-3` and 5`-CTACCTCCACCATGCCAAGTG-3`. VEGF release VEGF release was determined via Multi-Analyte Profiling by Myriad RBM (Austin, USA-TX) using the Human AngiogenesisMAP®. RESULTS After the hardware had returned to our laboratory, the evaluation of the samples was started. 5 of the 6 hardware units worked well during the mission. The cells in the last unit were not automatically fixed. Optical analysis confirmed the formation of MCS in all the units. Cells were harvested from the cultivation chambers (Fig. 2A). Supernatants were collected from the reserve tanks to determine protein release (Fig. 2B). MAP analysis identified a higher release of VEGF in the Space samples compared to ground controls. On the RPM VEGF release was reduced after 5 and 10d (Fig. 2C). To determine the source of VEGF release we further investigated gene expression via qPCR. Compared to 1g controls VEGFA transcription was down-regulated after 5d and strong down-regulated after 10d in r-µg, independently of 3D growth (Fig. 2D). In s-µg VEGFA transcription was only minimal reduced (Fig. 2E) with small differences between adherent cells and MCS. DISCUSSION The CellBox-2 mission was successful. The cells stayed alive in the flight hardware and we were able to isolate mRNA and protein from adherently growing cells and MCS respectively. With our focus on VEGF gene expression and protein release, because their alteration could trigger two further changes: The paracrine stimulation of angiogenesis, and the autocrine stimulation of the cancer cells themselves (Liu et al., 1995; Lichtenberger et al., 2010). We found a rather constant VEGFA expression under s-µg and a down-regulation in r-µg, that seemed not to be a feedback of MCS formation. The hypothesis is consistent with results of previous long-term studies on the RPM and from the Shenzhou-8/SIMBOX Space mission (Ma et al., 2014; Kopp et al., 2015) and may contribute to a less aggressive phenotype of FTC-133 cells cultured in µg (Ma et al., 2014). The MAP result however showed differences in the secretion behaviour. The VEGF release was increased under r-µg and decreased under s-µg. Reasons could be different materials of the cell chambers, as well as effects of shear forces on the RPM or of radiation in Space. Overall a higher release of VEGF may indicate a greater angiogenic potential of FTC-133 cells in Space. CONCLUSION & OUTLOOK During a Space experiment onboard the ISS, we found spheroid formation by human FTC-133 cells like during previous experiments using the RPM. Beyond the morphological similarities a comparison of VEGF mRNA expression and protein release indicated differences, when human FTC-133 cells were cultured in Space or incubated under s-µg. Future genome and proteome analyses may uncover further differences but also similarities. We expect to detect signalling pathways related to different biological processes, such as spheroid formation in vitro and metastasis in vivo. In the end, the detection of new gravisensitive proteins may be helpful to find new targets for cancer treatment. LEGENDS TO THE FIGURES Figure 1 | Flight hardware for the CellBox-2 experiment. (A) Schematic drawing of an automated hardware unit showing the main components. (B) Filling of medium and cells into the cell cultivation chamber. (C) The assembled hardware unit. (D) The complete set of 6 hardware units for the space experiment. (E) Fixation of the cells inside the hardware unit after 10d under 1g conditions (ground control). The pump is injecting RNAlater (white fluid from the right) in the cell cultivation chamber and simultaneously removing old medium (yellowish, on the left). Figure 2 | VEGF expression of FTC-133 cells cultured in µg. (A) Cell harvesting from the cultivation chamber of one hardware unit. (B) Collecting the supernatants from the reserve tank. (C) Changes in VEGF release after 5 and 10d in µg. (D) VEGFA expression in cells exposed to r-µg in Space or (E) exposed to s-µg on the RPM.