Abstract
We studied the behavior of Ewing’s Sarcoma cells of the line A673 under simulated microgravity (s-µg). These cells express two prominent markers—the oncogene EWS/FLI1 and the chemokine receptor CXCR4, which is used as a target of treatment in several types of cancer. The cells were exposed to s-µg in a random-positioning machine (RPM) for 24 h in the absence and presence of the CXCR4 inhibitor AMD3100. Then, their morphology and cytoskeleton were examined. The expression of selected mutually interacting genes was measured by qRT-PCR and protein accumulation was determined by western blotting. After 24 h incubation on the RPM, a splitting of the A673 cell population in adherent and spheroid cells was observed. Compared to 1 g control cells, EWS/FLI1 was significantly upregulated in the adherent cells and in the spheroids, while CXCR4 and CD44 expression were significantly enhanced in spheroids only. Transcription of CAV-1 was upregulated and DKK2 and VEGF-A were down-regulated in both, adherent in spheroid cells, respectively. Regarding, protein accumulation EWS/FLI1 was enhanced in adherent cells only, but CD44 decreased in spheroids and adherent cells. Inhibition of CXCR4 did not change spheroid count, or structure. Under s-µg, the tumor marker EWS/FLI1 is intensified, while targeting CXCR4, which influences adhesion proteins, did not affect spheroid formation.
Highlights
Ewing’s sarcoma (ES) is the s most common bone tumor in adolescents and young adults, accounting for approximately 3–5% of all pediatric tumors [1,2]
Simulated microgravity via random positioning machine (RPM) involves unique changes in cellular adhesion patterns: The loss of adherence to a cell culture flask of a fraction of formerly firmly attached cells and their reassembly to multicellular tumor spheroids (MCTS) thereby switching from two-dimensional adherence to a polystyrene surface to intercellular three-dimensional adherence without undergoing anoikis, while another fraction remains attached under the same circumstances, makes it a intriguing in vitro cancer model
We observed significantly upregulated gene expression and significantly increased protein accumulation of EWS/FLI1 at least in the adherent cells under s-μg
Summary
Ewing’s sarcoma (ES) is the s most common bone tumor in adolescents and young adults, accounting for approximately 3–5% of all pediatric tumors [1,2]. Simulated microgravity via RPM involves unique changes in cellular adhesion patterns: The loss of adherence to a cell culture flask of a fraction of formerly firmly attached cells and their reassembly to MCTS thereby switching from two-dimensional adherence to a polystyrene surface to intercellular three-dimensional adherence without undergoing anoikis, while another fraction remains attached under the same circumstances, makes it a intriguing in vitro cancer model Such a model could help identify molecular switches of adhesion pattern alterations and gain valuable insights in the dynamics of ES cell aggregation and –adherence, which could be of value to microgravity, but to cancer researchers in general. A better understanding of the processes involved in self-aggregation of certain tumor cells to a three-dimensional spheroid, while other cells remain attached under the same circumstances is certainly beneficial to microgravity researchers but cancer researchers as well and could expand our horizon on characteristics of tumor cell adherence and their behavior in in vitro three-dimensionality [56] and could potentially contribute to new therapeutic approaches or targets
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