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 [...].

We would like to thank Bevelacqua et al. for their interest in our article [1] and their comments [2]. We completely agree with the authors that the complex space environment cannot be adequately simulated on Earth. However, this was not our intention for these studies on tumor spheroids. The influence of space radiation would even be counterproductive in targeting a metastasis model to reflect the situation in cancer patients on Earth.

For many years, cells have been exposed to conditions of microgravity and scientists have investigated how these cells might sense or adapt to microgravity [3,4]. Growing three-dimensional cell aggregates in space and in laboratories using microgravity simulators is a typical application of microgravity research [5]. The NASA-developed rotating wall vessel bioreactor was specially applied to engineer such tissue constructs. The random positioning machine (RPM) works as a three-dimensional clinostat negating the directional influence of the g-vector providing simulated conditions of micro- or partial gravity (also referred as “time-averaged microgravity” by some authors). It should be noted and we are aware that simulated microgravity differs from real (space) microgravity in several aspects. Nevertheless, the RPM has become a well-established device for gravitational research [6,7], which is also recommended by international space agencies (like NASA [8] or the European Space Agency, ESA [9]) for experiments in reduced gravity. Although it is known to produce additional shear stress and fluid dynamics within the cell culture flasks [10,11], Wuest et al. also evaluated the RPM as a “reliable tool supporting ground-based microgravity studies” and an “ideal tool for preliminary microgravity tests, screening studies in which simulated microgravity effects are checked on various organisms” [12]. This is caused by the fact that cellular effects that were observed in real microgravity could be reproduced with good agreement on RPMs [13,14,15,16,17,18]. In this study, however, these questions were not of importance, as the RPM merely served as a tool for the generation of tumor spheroids. We believe that the RPM is superior to other methods of spheroid generation such as liquid overlay techniques or spinner flasks, as in this system it is possible to induce cells to detach from an already established cellular network and to form spheroids suspended in the culture medium. These spheroids, which were also found after long-term cultures in space [19], are an important model system to mimic micrometastases or microregions of solid tumors [20]. Despite their simplicity, tumor spheroids are often used for drug testing in pharmacology today [21,22,23] and are still state of the art. More complex tumor organoids co-cultured with different cell types are in development and will replace the homogenous spheroids in the future, better mimicking tumor biology in vivo. In addition, the investigation of microgravity-induced cell detachment and spheroid formation delivers valuable information about processes like metastasis and in vivo cancer progression [22]. The RPM-based metastasis model can be effectively used to study in vitro whether drugs can favor or inhibit spheroid formation. Of course, mechanical forces have to be taken into consideration while designing experiments with the RPM and analyzing their results. However, this can be done by using appropriate controls. Moreover, cells inside the human body, especially those in the process of forming metastases, are subjected to a multitude of forces besides gravity; therefore, the implementation of a completely shear-force-free experimental environment would not improve the results.

In summary, the arguments of Bevelacqua et al. [2] may result from misunderstandings of the actual purpose of the experiments. Not only has it never been the intention of this study to simulate space conditions in complete detail, it was never intended to simulate space at all and we never claimed to do so. Our aim was to investigate molecular mechanisms of metastasis development and the identification of possible target molecules for potential antimetastatic pharmacological interventions using a device that, by simulating certain aspects of microgravity, induces the formation of spheroids from originally adherently growing tumor cells, something that cannot be achieved as easily by other established methods. Some years ago Becker and Souza wrote: “Combination of the resources available in the unique environment of microgravity with the tools and advanced technologies that exist in laboratories across Earth may inform new research approaches to expand the knowledge necessary for improving treatment options, and enhancing the quality of life for those affected by this illness.” [24]. In our study, we used space-derived investigations to fight cancer on Earth.

We thank the editors for giving us the opportunity to provide a reply to the letter.