Several years ago, we detected the formation of multicellular spheroids in experiments with human thyroid cancer cells cultured on the Random Positioning Machine (RPM), a ground-based model to simulate microgravity by continuously changing the orientation of samples. Since then, we have studied cellular mechanisms triggering the cells to leave a monolayer and aggregate to spheroids. Our work focused on spheroid-related changes in gene expression patterns, in protein concentrations, and in factors secreted to the culture supernatant during the period when growth is altered. We detected that factors inducing angiogenesis, the composition of integrins, the density of the cell monolayer exposed to microgravity, the enhanced production of caveolin-1, and the nuclear factor kappa B p65 could play a role during spheroid formation in thyroid cancer cells. In this study, we performed a deep proteome analysis on FTC-133 thyroid cancer cells cultured under conditions designed to encourage or discourage spheroid formation. The experiments revealed more than 5900 proteins. Their evaluation confirmed and explained the observations mentioned above. In addition, we learned that FTC-133 cells growing in monolayers or in spheroids after RPM-exposure incorporate vinculin, paxillin, focal adhesion kinase 1, and adenine diphosphate (ADP)-ribosylation factor 6 in different ways into the focal adhesion complex.

Human MCF-7 breast cancer cells were exposed to a Random Positioning Machine (RPM). After 24 hours (h) the cells grew either adherently within a monolayer (AD) or within multicellular spheroids (MCS). AD and MCS populations were separately harvested, their cellular differences were determined performing qPCR on genes, which were differently expressed in AD and MCS cells. Gene array technology was applied to detect RPM-sensitive genes in MCF-7 cells after 24 h. Furthermore, the capability to form multicellular spheroids in vitro was compared with the intracellular distribution of NF-kappaB (NFκB) p65. NFκB was equally distributed in static control cells, but predominantly localized in the cytoplasm in AD cells and nucleus in MCS cells exposed to the RPM. Gene array analyses revealed a more than 2-fold change of only 23 genes including some whose products are affected by oxygen levels or regulate glycolysis. Significant upregulations of the mRNAs of enzymes degrading heme, of ANXA1, ANXA2, CTGF, CAV2 and ICAM1, as well as of FAS, Casp8, BAX, p53, CYC1 and PARP1 were observed in MCS cells as compared with 1g-control and AD cells. An interaction analysis of 47 investigated genes suggested that HMOX-1 and NFκB variants are activated, when multicellular spheroids are formed.

SummaryBackground: For tendon tissue engineering, tenocyte-seeded scaffolds are a promising approach. Under conventional 2D culture however, tenocytes show rapid senescene and phenotype loss. We hypothesized that phenotype loss could be counteracted by simulated microgravity conditions. Methods: Human tenocytes were exposed to microgravity for 9 days on a Random Positioning Machine (RPM). Formation of 3D-structures (spheroids) was observed under light microscopy, gene expression was measured by realtime PCR. Cells under conventional 2D-culture served as control group. Results: Simulated microgravity reached a value of as low as 0.003g. Spheroid formation was observed after 4 days, and spheroids showed stable existance to the end of the observation period. After 9 days, spheroids showed a significantly higher gene expression of collagen 1 (Col1A1) compared to adherent cells under microgravity (4.4x, p=0.04) and compared to the control group (5.6x, p=0.02). Gene expression of collagen 3 (COL3A1) was significantly increased in spheroids compared to the control group (2.3x, p=0.03). Gene expressions of the extracellular matrix genes Tenascin C und Fibronectin (TNC and FN) were increased in adherent cells under microgravity compared to the 1g-control group, not reaching statistical significance (p=0.1 and p=0.3). For the gene expression of vimentin, no significant alteration was observed both in the adherent cells and in the spheroids compared to the 1g control group. Gene expression of the tenocyte-specific transcription factor scleraxis (SCX) was significantly increased in spheroids compared to the control group (3.7x, p=0.03). Conclusion: Simulated microgravity could counteract tenocyte senescence in vitro and serve as a promising model for scaffold-free 3D cell culturing and tissue engineering. Level of evidence: V (laboratory study).

VEGF enhances proliferation and expression of tendon-related genes in human tenocytes. It could therefore be a useful addition for tenocyte cultivation.