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Spheroid formation of human thyroid cancer cells under simulated microgravity: a possible role of CTGF and CAV1.

Warnke E, Pietsch J, Wehland M, Bauer J, Infanger M, Görög M, Hemmersbach R, Braun M, Ma X, Sahana J, Grimm D - Cell Commun. Signal (2014)

Bottom Line: However, each GBF might exert device-specific and altered superimposingly gravity-dependent effects on the cells.A decreased expression of CAV1 and CTGF in MCTS compared to adherent cells was observed after cultivation on both machines.A relationship between these molecules and MCTS formation is discussed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Clinic for Plastic, Aesthetic and Hand Surgery, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany. daniela.grimm@farm.au.dk.

ABSTRACT

Background: Multicellular tumor spheroids (MCTS) formed scaffold-free under microgravity are of high interest for research and medicine. Their formation mechanism can be studied in space in real microgravity or on Earth using ground-based facilities (GBF), which simulate microgravity. On Earth, these experiments are more cost-efficient and easily performable. However, each GBF might exert device-specific and altered superimposingly gravity-dependent effects on the cells.

Results: FTC-133 human thyroid cancer cells were cultivated on a 2D clinostat (CN) and a random positioning machine (RPM) and compared with corresponding 1 g control cells. Harvested cell samples were investigated by microscopy, quantitative realtime-PCR and Multi-Analyte Profiling. Spheroid formation and growth occurred during 72 h of cultivation on both devices. Cytokine secretion and gene activation patterns frequently altered in different ways, when the cells were cultured either on the RPM or the CN. A decreased expression of CAV1 and CTGF in MCTS compared to adherent cells was observed after cultivation on both machines.

Conclusion: The development of MCTS proceeds similarly on the RPM and the CN resembling the situation observed under real microgravity conditions, while no MCTS formation was observed at 1 g under identical experimental conditions. Simultaneously, changes in the regulation of CTGF and CAV1 appeared in a comparable manner on both machines. A relationship between these molecules and MCTS formation is discussed.

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Quantitative real-time PCR for the determination of alterations in gene-expression of selected genes after 72 h. CAV1 (A, B), CAV2 (C, D), CTGF (E, F), EGF (G, H), ERK1 (I, J), IL-8 (K, L), ITGB1 (M, N), and PRKCA (O, P) gene expression was analyzed after 72 h exposure of the cells to 2-D Clinostat (A, C, E, G, I, K, M, O) or random positioning machine (RPM; B, D, F, H, J, L, N, P). After 72 h culturing, the FTC-133 cells grew adherently (AD) or within the MCTS. On both machines CAV1 (C, D) was down-regulated in the MCTS cells and CTGF (E, F) was differently expressed in AD and MCTS, respectively. All results are shown as mean ± standard deviation (SD) of n = 10 independent samples, with significance indicated by *P < 0.05 vs. 1g, **P < 0.05 vs. s-μg: AD.
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Figure 3: Quantitative real-time PCR for the determination of alterations in gene-expression of selected genes after 72 h. CAV1 (A, B), CAV2 (C, D), CTGF (E, F), EGF (G, H), ERK1 (I, J), IL-8 (K, L), ITGB1 (M, N), and PRKCA (O, P) gene expression was analyzed after 72 h exposure of the cells to 2-D Clinostat (A, C, E, G, I, K, M, O) or random positioning machine (RPM; B, D, F, H, J, L, N, P). After 72 h culturing, the FTC-133 cells grew adherently (AD) or within the MCTS. On both machines CAV1 (C, D) was down-regulated in the MCTS cells and CTGF (E, F) was differently expressed in AD and MCTS, respectively. All results are shown as mean ± standard deviation (SD) of n = 10 independent samples, with significance indicated by *P < 0.05 vs. 1g, **P < 0.05 vs. s-μg: AD.

Mentions: After 72 h of exposure to the CN or the RPM, cells had parted into adherent cells (AD) and cells forming multicellular tumor spheroids (MCTS), which floated in the supernatant (Figure 1 I). At this time, two fractions were harvested from the CN and the RPM, respectively. Subsequently, it was investigated how the expression of the selected genes (Table 1) had been regulated within the 4 cell samples indicated in Figure 3 as compared to the corresponding static ground controls (1 g). Interestingly, the regulation of CTGF and CAV1 split on both machines equally. In adherent cells, CTGF remained up-regulated and CAV1 unchanged, while in MCTS the expression of these two genes was decreased (Figure 3 E-F). Other genes have also changed their expression behavior, but differently, when incubated on the two machines. Exposure of FTC-133 cells to the RPM led to an up-regulation of ERK1 and EGF gene-expression in MCTS, but did not affect one of the selected genes in AD cells. In contrast, exposure of the same type of cells to CN triggered down-regulation of ERK1, ITGB1, and PRKCA gene-expression in MCTS only (Figure 3 I, M, O), but decreased expression of CAV2 and IL8 in both, AD and MCTS cells (Figure 3 C, K).


Spheroid formation of human thyroid cancer cells under simulated microgravity: a possible role of CTGF and CAV1.

Warnke E, Pietsch J, Wehland M, Bauer J, Infanger M, Görög M, Hemmersbach R, Braun M, Ma X, Sahana J, Grimm D - Cell Commun. Signal (2014)

Quantitative real-time PCR for the determination of alterations in gene-expression of selected genes after 72 h. CAV1 (A, B), CAV2 (C, D), CTGF (E, F), EGF (G, H), ERK1 (I, J), IL-8 (K, L), ITGB1 (M, N), and PRKCA (O, P) gene expression was analyzed after 72 h exposure of the cells to 2-D Clinostat (A, C, E, G, I, K, M, O) or random positioning machine (RPM; B, D, F, H, J, L, N, P). After 72 h culturing, the FTC-133 cells grew adherently (AD) or within the MCTS. On both machines CAV1 (C, D) was down-regulated in the MCTS cells and CTGF (E, F) was differently expressed in AD and MCTS, respectively. All results are shown as mean ± standard deviation (SD) of n = 10 independent samples, with significance indicated by *P < 0.05 vs. 1g, **P < 0.05 vs. s-μg: AD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4020378&req=5

Figure 3: Quantitative real-time PCR for the determination of alterations in gene-expression of selected genes after 72 h. CAV1 (A, B), CAV2 (C, D), CTGF (E, F), EGF (G, H), ERK1 (I, J), IL-8 (K, L), ITGB1 (M, N), and PRKCA (O, P) gene expression was analyzed after 72 h exposure of the cells to 2-D Clinostat (A, C, E, G, I, K, M, O) or random positioning machine (RPM; B, D, F, H, J, L, N, P). After 72 h culturing, the FTC-133 cells grew adherently (AD) or within the MCTS. On both machines CAV1 (C, D) was down-regulated in the MCTS cells and CTGF (E, F) was differently expressed in AD and MCTS, respectively. All results are shown as mean ± standard deviation (SD) of n = 10 independent samples, with significance indicated by *P < 0.05 vs. 1g, **P < 0.05 vs. s-μg: AD.
Mentions: After 72 h of exposure to the CN or the RPM, cells had parted into adherent cells (AD) and cells forming multicellular tumor spheroids (MCTS), which floated in the supernatant (Figure 1 I). At this time, two fractions were harvested from the CN and the RPM, respectively. Subsequently, it was investigated how the expression of the selected genes (Table 1) had been regulated within the 4 cell samples indicated in Figure 3 as compared to the corresponding static ground controls (1 g). Interestingly, the regulation of CTGF and CAV1 split on both machines equally. In adherent cells, CTGF remained up-regulated and CAV1 unchanged, while in MCTS the expression of these two genes was decreased (Figure 3 E-F). Other genes have also changed their expression behavior, but differently, when incubated on the two machines. Exposure of FTC-133 cells to the RPM led to an up-regulation of ERK1 and EGF gene-expression in MCTS, but did not affect one of the selected genes in AD cells. In contrast, exposure of the same type of cells to CN triggered down-regulation of ERK1, ITGB1, and PRKCA gene-expression in MCTS only (Figure 3 I, M, O), but decreased expression of CAV2 and IL8 in both, AD and MCTS cells (Figure 3 C, K).

Bottom Line: However, each GBF might exert device-specific and altered superimposingly gravity-dependent effects on the cells.A decreased expression of CAV1 and CTGF in MCTS compared to adherent cells was observed after cultivation on both machines.A relationship between these molecules and MCTS formation is discussed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Clinic for Plastic, Aesthetic and Hand Surgery, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany. daniela.grimm@farm.au.dk.

ABSTRACT

Background: Multicellular tumor spheroids (MCTS) formed scaffold-free under microgravity are of high interest for research and medicine. Their formation mechanism can be studied in space in real microgravity or on Earth using ground-based facilities (GBF), which simulate microgravity. On Earth, these experiments are more cost-efficient and easily performable. However, each GBF might exert device-specific and altered superimposingly gravity-dependent effects on the cells.

Results: FTC-133 human thyroid cancer cells were cultivated on a 2D clinostat (CN) and a random positioning machine (RPM) and compared with corresponding 1 g control cells. Harvested cell samples were investigated by microscopy, quantitative realtime-PCR and Multi-Analyte Profiling. Spheroid formation and growth occurred during 72 h of cultivation on both devices. Cytokine secretion and gene activation patterns frequently altered in different ways, when the cells were cultured either on the RPM or the CN. A decreased expression of CAV1 and CTGF in MCTS compared to adherent cells was observed after cultivation on both machines.

Conclusion: The development of MCTS proceeds similarly on the RPM and the CN resembling the situation observed under real microgravity conditions, while no MCTS formation was observed at 1 g under identical experimental conditions. Simultaneously, changes in the regulation of CTGF and CAV1 appeared in a comparable manner on both machines. A relationship between these molecules and MCTS formation is discussed.

Show MeSH
Related in: MedlinePlus