Microgravity promotes gap junctional contact formation in TCam-2 seminoma cells grown in low adhesion as embryoid bodies.

The study of how mechanical forces may influence cell behavior is a relevant challenge of nowadays that may allow us to define the relationship between mechanics and biochemistry. Literature data reported that microgravity alters cell behavior through cytoskeleton and/or junction modifications. It has been already reported, by our group, that TCam-2 seminoma cells are sensible to microgravity when cultured in adhesion on plastic or glass dishes (Ferranti et al., 2014, Biomed. Res. Int.). However, TCam-2 cells are able to grow also when cultured in low adhesion conditions, forming floating embryoid bodies (EB). This study has the aim to verify if microgravity can affect TCam-2 cell behavior even in absence of cell-substrate adhesion. We investigated the morphological, cytoskeletal and junctional features of TCam-2 cell EB cultured for 24 hours in control and simulated microgravity conditions. Simulated microgravity was obtained using the Random Positioning Machine (RPM; Dutch Space, Netherlands). We observed by TEM, that junctional capability of TCam-2 EB, changes in microgravity condition, being RPM cultured EB involved in gap junctional contacts that do not form in control condition. Noteworthy, gap junction loss is related to seminoma neoplastic progression, and seminoma samples are characterized by an aberrant connexin-43 (CX-43) localization in the Golgi apparatus (Roger et al., 2004, J Pathol; Brehm et al., 2006, Neoplasia). Interestingly, we observed that simulated microgravity-exposed samples show a strong CX-43 membranous staining, whereas, consistent to literature data, a CX-43 low positivity is observable only in the cytoplasm of control cells. In addition, we found that microgravity also affects TCam-2 microtubule organization, strongly indicating that microtubule-dependent membrane trafficking changes in RPM cultured cells. All together these data indicate that TCam-2 cell are sensitive to gravity force changes even in absence of cell-substrate adhesion.