Abstract
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.
Highlights
Multicellular spheroids (MCS) are interesting models of cancer [1,2]
Spheroids may be generated in various ways [1]. One example is their exposure to real (r-μg) and simulated microgravity (s-μg) [3]. s-μg can be achieved by application of the Random Positioning Machine (RPM), a device created to simulate microgravity on Earth
These efforts revealed that exposure of FTC-133 cells to microgravity enhances apoptosis and promotes nuclear factor kappa B (NFκB) p65 activities, while the caveolin 1 (CAV-1) gene is down-regulated during spheroid formation [6,10,11,12]
Summary
Multicellular spheroids (MCS) are interesting models of cancer [1,2]. They resemble natural tumors more than cell monolayers, but they are not as complex as those [3]. Spheroid formation under microgravity in vitro includes a cell leaving a two-dimensional monolayer and joining neighbor cells in a three-dimensional manner This process is supposed to mirror, in part, the change of a cancer cell’s kind of growth in vivo as it is observed during metastasis [4]. The harvested samples were analyzed by applying microscopy, gene array technology, quantitative real time-PCR, and Multi-Analyte Profiling with the aim to examine cell morphologies, gene expression patterns, and proteins which could be associated with the transition from a two- to a three-dimensional growth pattern [5,6,8,9] Taken together, these efforts revealed that exposure of FTC-133 cells to microgravity enhances apoptosis and promotes nuclear factor kappa B (NFκB) p65 activities, while the caveolin 1 (CAV-1) gene is down-regulated during spheroid formation [6,10,11,12]. Sample Number Pre-incubation Following 3 days incubation under . . . Kind of growth at time of harvest Number of proteins detected
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