One of the hallmarks of microgravity-induced effects in several cellular models is represented by the alteration of oxidative balance with the consequent accumulation of Reactive Oxygen Species (ROS). It is well known that male germ cells are sensitive to oxidative stress and to changes of gravitational force even if published data on germ cell models are scarce. To gain more insights into the mechanisms of male germ cell response to altered gravity, a 3D cell culture model has been established from TCam-2 cells, a seminoma-derived cell line considered the only human cell line available to study in vitro mitotically active human male germ cells. TCam-2 cell spheroids were cultured for 24 hours under unitary gravity (Ctr) or simulated microgravity (s-microgravity) conditions, these last ones were obtained using the Random Positioning Machine (RPM). A significant increase in intracellular ROS and mitochondria superoxide anion levels has been observed after RPM exposure. In line with these results a trend of protein and lipid oxidation increase, and increased pCAMKII expression levels were observed after RPM exposure. The ultrastructural analysis by Transmission Electron Microscopy revealed that RPM-exposed mitochondria appeared enlarged and, even if seldom, disrupted. Notably, even the expression of the main enzymes involved in the redox homeostasis appears modulated by RPM exposure in a compensatory way, being GPX1, NCF1, and CYBB downregulated, whereas NOX4 and HMOX1 upregulated. Interestingly, HMOX1 is involved in the heme catabolism of mitochondria cytochromes, and therefore the positive modulation of this marker can be associated to the observed mitochondria alteration. All together, these data demonstrate TCam-2 spheroid sensitivity to acute SM exposure and indicate the capability of these cells to trigger compensatory mechanisms that allow to overcome the exposure to altered gravitational force.

The early detection of cutaneous melanoma, a potentially lethal cancer with rising incidence, is key to increase survival and therapeutic adjustment. Especially in stages II-IV biomarkers are urgently needed for adjuvant therapy purposes after resection and for treatment of metastatic patients. We here investigated if fatty acid (FA) and protein composition of small extracellular vesicles (sEV) deriving from plasma of 0-I, II, and III-IV stage melanoma patients (n=38) could reflect disease stage and thus function as biomarker. The subpopulation of sEV expressing CD81 (CD81sEV) was isolated by an ad hoc immune affinity technique from microvesicle-depleted plasma. Biological macromolecules were investigated by gas chromatography and mass spectrometry in CD81sEV. A higher content of FA and a decrease in Saturation Index (C18:0/C18:1), already detectable in early stages, distinguished patients’ from healthy donor CD81sEV. Proteomics (identifier PXD024434) detected an exclusive and significant increase of CD14, PON1, PON3 and APOA5 in stage II and a significant decrease of Rap1b in stage III-IV CD81sEV. The FA and proteomic stage dependent CD81sEV signature strengthens the potential of circulating sEV studies in providing discriminatory information for early diagnosis, prediction of metastatic behavior and follow up of melanoma patients.