Event Abstract

Back to Event

Alterations of the Cytoskeleton in Breast Cancer Cells during Microgravity visualised by FLUMIAS Live-Cell Imaging

Mohamed Z. Nassef1, Sascha Kopp1, Daniela Melnik1, Marcus Krüger1*, Markus Wehland1, Thomas J. Corydon2, Johann Bauer3, Manfred Infanger1 and Daniela Grimm1, 2
  • 1 Medizinische Fakultät, Universitätsklinikum Magdeburg, Germany
  • 2 Aarhus University, Denmark
  • 3 Max-Planck-Institut für Biochemie, Germany

ABSTRACT The cytoskeleton is a highly dynamic structure playing an important role in graviperception and gravisensitivity of cells exposed to microgravity. The cytoskeletal network is closely connected to many cellular processes and functions. The exact mechanisms are mostly unknown. For the first time, we investigated human MCF-7 breast cancer cells in real microgravity. We were particularly interested in studying early-stage cytoskeletal changes (actin, microtubules) and early mechanisms of tumour spheroid formation. The current study was performed during the TEXUS-54 sounding rocket flight using live-cell imaging of modified MCF-7 cells simultaneously expressing LifeAct-GFP and mCherry-tubulin marker proteins and the FLUMIAS confocal laser spinning disc fluorescence microscope. We detected early alterations of the cytoskeleton during the cells’ exposure to microgravity, including disturbance of F-actin bundles and appearance of filopodia- and lamellipodia-like structures. The MCF-7 cells reacted to microgravity within four minutes and showed similar alterations compared to FTC-133 thyroid cancer cells that were investigated before. This might indicate a common graviperception in these cancer cells. INTRODUCTION Astronauts, who spend a long time in Space, suffer from several side effects such as cardiovascular problems, bone loss and immune system alterations (White and Averner, 2001). The cause of these side effects are changes at the cellular level such as an altered cellular function and morphology together with activated molecular mechanisms as a response of the cells to microgravity (µg). Space research has also attracted the attention of cancer biologists who search for new targets for therapies (Becker and Souza, 2013). In order to unravel the intracellular modifications that happen in real µg (r-µg), we performed several parabolic flights and sounding rocket missions with human cancer cells (Ma et al., 2014; Corydon et al., 2016; Krüger et al., 2017). The human breast cancer cell line MCF-7 cells had been extensively studied under simulated µg (s-µg) conditions using the RPM (Kopp et al., 2016; Kopp et al., 2018; Sahana et al., 2018). In addition, these cells are very robust, when cultured under µg especially in Space (Vassy et al., 2001) or during a parabolic flight (unpublished results). As the cytoskeleton is assumed to act as a gravisensor in eukaryotic cells (Hughes-Fulford, 2003), we intend to investigate the effect of µg on cytoskeletal rearrangements of human MCF-7 breast cancer cells during the TEXUS („Technologische Experimente unter Schwerelosigkeit“ (TX))-54 sounding rocket flight by means of live-cell imaging. MATERIALS AND METHODS Cell culture MCF-7 cells were cultured as previously described (Kopp et al., 2018). In addition to 10% FCS and 1% penicillin/streptomycin, G418 was added to allow growth of the transfected cells only. Construction of an expression cassette to visualize F-actin and 𝛼-tubulin The expression construct was produced as published previously (Corydon et al., 2016). It is demonstrated in Fig. 1A. Generation of MCF-7 cells expressing LifeAct-eGFP-IRES-mCherry-Tubulin The MCF-7 cell line was stably transfected using a Sleeping Beauty Transposon-based vector containing the LifeAct-eGFP-IRES-mCherry-Tubulin (LAGICT) expression cassette for the visualization of F-actin and 𝛼-tubulin (Aronovich et al., 2011; Corydon et al., 2016). TEXUS-54 sounding rocket mission The sounding rocket mission was performed as previously described (Corydon et al., 2016). Live-cell imaging by the FLUMIAS Microscope Approximately 7000 MCF-7 cells were seeded into one channel of an Ibidi ibiTreat µ-slide VI 0.4 (Fig. 1B). The slide was temperature controlled and loaded into the FLUMIAS microscope (Corydon et al., 2016) shortly before the launch. Five minutes prior to launch three z-stacks were obtained from pre-selected cells as a ground control. About 75s after launch the µg phase was reached, and the microscope started recording the pre-selected cells Three z-stacks were taken every one minute with 125ms exposure time. The thickness of the z-stack was 21µm with 0.5µm step size. The procedure was repeated four times with a total number of five active phases covering 6min of µg. After recovery of the image data, a single image was extracted from each z-stack taken during µg. The extracted images were deconvolved by Huygens Essential Scientific Volume Imaging software and compared to a control image taken on ground. RESULTS To increase the current knowledge about cytoskeletal alterations of human breast cancer cells exposed to r-µg conditions, live-cell imaging was performed using MCF-7 cells during a sounding rocket mission. The TX-54 mission was performed in May 2018 at Esrange Space Center, Kiruna, Sweden. We investigated MCF-7 cells which were transfected with LAGICT expression cassette for visualization of F-actin and α-tubulin. The observation was performed and recorded using the FLUMIAS microscope onboard the TX sounding rocket. The microscopic images were analysed to evaluate the effect of µg on the MCF-7 cytoskeleton. We detected F-actin changes such as disturbances of F-actin bundles and the appearance of filopodia- and lamellipodia-like structures (Fig. 2, white and yellow arrows). In breast cancer cells no morphological signs of programmed cell death were detectable. DISCUSSION For the first time, we were able to show the effect of r-µg on the cytoskeletal structure of breast cancer cells during a sounding rocket mission in real time. The images obtained from the FLUMIAS microscope during the TX-54 mission were compared to those from the TX-52 mission (Corydon et al., 2016), which was performed with FTC-133 follicular thyroid cancer cells. Both missions showed similar effects of r-µg on the two different cell lines. This is a further prove that the cytoskeleton is affected by µg very early (Grosse et al., 2012) and enforces the suggestion that the cytoskeleton directly senses µg (Vorselen et al., 2014). CONCLUSION & OUTLOOK In order to further confirm the hypothesis of direct sensing of µg by the cytoskeleton, our next step is to investigate the gene activation status of MCF-7 after short time exposure of the cells to r-µg. In this way we intend to uncover signalling pathways that are connected with extracellular matrix, cell adhesion, cytoskeleton, cytokines, growth factors, cell cycle and apoptosis. This should enable us to learn the molecular mechanisms for initial sensing and adaptation to µg of breast cancer cells. LEGENDS TO THE FIGURES Figure 1 | (A) Diagram showing the transfection of MCF-7 cells with LifeAct-eGFP-IRES-mCherry-Tubulin (LAGICT) expression cassette. Microscopic images showing visualization of F-Actin (488 laser) and α-tubulin (568 laser). (B) 18-well Ibidi slides used inside the FLUFIX (image from ibidi.com). (C) FLUFIX used to incubate 18-well Ibidi slides to be fixed during the TX-54 mission. (D) Ibidi µ-slide VI 0.4 ibiTreat prepared for the FLUMIAS microscope. (E) The late access and fixation unit after installing the Ibidi slide. (F) Image of TX-54 sounding rocket showing the accommodation of FLUFIX and FLUMIAS inside the rocket (courtesy of Airbus Defence & Space). Figure 2 | Timeline and images obtained by FLUMIAS showing the MCF-7 cells 5 min before the launch (T-300s) of the rocket and during the µg phase (T+150s–T+312s). White arrows indicate the changes occurring to the F-Actin and yellow arrows indicate the changes occurring to the α-tubulin.

Figure 1
Image
Figure 2
Image

Acknowledgements

This work was supported by the German Space Agency (DLR), BMWi project 50WB1524 (DG). We like to thank Dr. Markus Braun and Dr. Otfried Joop (German Space Agency, DLR), the engineers Andreas Schütte, Dr. Hergen Oltmann, Burkhard Schmitz, and Stefan Feldmann (Airbus Defence & Space) for their wonderful support of the TEXUS-54 mission.

References

Aronovich, E.L., McIvor, R.S., and Hackett, P.B. (2011). The Sleeping Beauty transposon system: a non-viral vector for gene therapy. Hum. Mol. Genet. 20(R1), R14-20. doi: 10.1093/hmg/ddr140. Becker, J.L., and Souza, G.R. (2013). Using space-based investigations to inform cancer research on Earth. Nat. Rev. Canc. 13, 315. doi: 10.1038/nrc3507. Corydon, T.J., Kopp, S., Wehland, M., Braun, M., Schütte, A., Mayer, T., et al. (2016). Alterations of the cytoskeleton in human cells in space proved by life-cell imaging. Sci. Rep. 6, 20043. doi: 10.1038/srep20043. Grosse, J., Wehland, M., Pietsch, J., Schulz, H., Saar, K., Hübner, N., et al. (2012). Gravity-sensitive signaling drives 3-dimensional formation of multicellular thyroid cancer spheroids. FASEB J. 26(12), 5124-5140. doi: 10.1096/fj.12-215749. Hughes-Fulford, M. (2003). Function of the cytoskeleton in gravisensing during spaceflight. Adv. Space Res. 32(8), 1585-1593. doi: 10.1016/s0273-1177(03)90399-1. Kopp, S., Sahana, J., Islam, T., Petersen, A.G., Bauer, J., Corydon, T.J., et al. (2018). The role of NFkappaB in spheroid formation of human breast cancer cells cultured on the Random Positioning Machine. Sci. Rep. 8(1), 921. doi: 10.1038/s41598-017-18556-8. Kopp, S., Slumstrup, L., Corydon, T.J., Sahana, J., Aleshcheva, G., Islam, T., et al. (2016). Identifications of novel mechanisms in breast cancer cells involving duct-like multicellular spheroid formation after exposure to the Random Positioning Machine. Sci. Rep. 6, 26887. doi: 10.1038/srep26887. Krüger, M., Wehland, M., Kopp, S., Corydon, T.J., Infanger, M., and Grimm, D. (2017). Life-Cell Imaging of F-Actin Changes Induced By 6 Min of Microgravity On A TEXUS Sounding Rocket Flight. Available online: http://pac.spaceflight.esa.int/. Proceedings of the 23rd ESA Symposium on European Rocket and Balloon Programmes and Related Research; 2017 Jun 11-15; Visby, Sweden. Ma, X., Pietsch, J., Wehland, M., Schulz, H., Saar, K., Hübner, N., et al. (2014). Differential gene expression profile and altered cytokine secretion of thyroid cancer cells in space. FASEB J. 28(2), 813-835. doi: 10.1096/fj.13-243287. Sahana, J., Nassef, M.Z., Wehland, M., Kopp, S., Krüger, M., Corydon, T.J., et al. (2018). Decreased E-Cadherin in MCF7 Human Breast Cancer Cells Forming Multicellular Spheroids Exposed to Simulated Microgravity. Proteomics 18(13), e1800015. doi: 10.1002/pmic.201800015. Vassy, J., Portet, S., Beil, M., Millot, G., Fauvel-Lafeve, F., Karniguian, A., et al. (2001). The effect of weightlessness on cytoskeleton architecture and proliferation of human breast cancer cell line MCF-7. FASEB J. 15(6), 1104-1106. Vorselen, D., Roos, W.H., MacKintosh, F.C., Wuite, G.J., and van Loon, J.J. (2014). The role of the cytoskeleton in sensing changes in gravity by nonspecialized cells. FASEB J. 28(2), 536-547. doi: 10.1096/fj.13-236356. White, R.J., and Averner, M. (2001). Humans in space. Nature 409(6823), 1115-1118. doi: 10.1038/35059243.

Keywords: Cytoskeleton, Actin, Tubulin, microgravity, Cancer, Sounding rocket, live-cell imaging

Conference: 39th ISGP Meeting & ESA Life Sciences Meeting, Noordwijk, Netherlands, 18 Jun - 22 Jun, 2018.

Presentation Type: Extended abstract

Topic: Biology and Cells Models

Citation: Nassef MZ, Kopp S, Melnik D, Krüger M, Wehland M, Corydon TJ, Bauer J, Infanger M and Grimm D (2019). Alterations of the Cytoskeleton in Breast Cancer Cells during Microgravity visualised by FLUMIAS Live-Cell Imaging. Front. Physiol. Conference Abstract: 39th ISGP Meeting & ESA Life Sciences Meeting. doi: 10.3389/conf.fphys.2018.26.00008

Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters.

The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated.

Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed.

For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions.

Received: 02 Dec 2018; Published Online: 16 Jan 2019.

* Correspondence: Dr. Marcus Krüger, Medizinische Fakultät, Universitätsklinikum Magdeburg, Magdeburg, Saxony-Anhalt, Germany, marcus.krueger@med.ovgu.de