Fig 1.
Spheroid generation is cell line dependent.
(A) 2,500 cells were seeded into 96-well plates (poly-HEMA coated). After 96h, total number of spheroids per well were counted. (B) 2 ×104cells/well were plated into 12-well culture plates and at indicated times cells were counted. The data points shown in (A) and (B) represent the mean values ± standard deviation (SD) of three independent experiments. (C) Cells were lysed, and protein samples were subjected to western blot analysis with specific antibodies against EGFR. GAPDH was used as a loading control.
Fig 2.
Autocrine EGFR signalling is maintained in spheroid culture of HNSCC cell lines.
Cells were cultured as monolayer (M) or spheroids (FS) for 24h, 48h or 72h in serum-free medium. The expression levels of pERK1/2 (42/44 kDa) and vinculin (internal loading control; 124 kDa) were analysed by immunoblotting. The values of the quantitative analysis are depicted as pERK1/2 protein levels relative to vinculin expression levels.
Fig 3.
Effects of EGFR activation/blockade on spheroid formation.
Cells were seeded at a density of 300,000 cells/well into 6-well plates and cultured under non-adherent conditions in the absence or presence of EGFR ligands (AREG, EGF) or EGFR blocking agents (CTX, gefitinib). Pictures were taken 72h after cell seeding. (5x magnification)
Fig 4.
Effects of EGFR activation/blockade on spheroid volume.
Cells were seeded at a density of 300,000 cells/well into poly-HEMA coated 6-well plates and treated with the indicated agents. After 72h, the diameters of at least 15 spheroids were measured and spheroid volumes calculated relative to the untreated spheroids. Bars represent mean values ± SD of four independent experiments. For the cell line SCC-9 the y-axis was adapted due to the EGF-induced strong increase in spheroid volumes.
Fig 5.
Activation of EGFR protects HNSCC cells against anoikis.
Cells were seeded at a density of 300,000 cells/well in non-coated or poly-HEMA coated 6-well plates and were immediately treated with either EGFR ligands or blocking reagents. After 72h, cells from monolayer (M) and forced suspension (FS) cultures were harvested and the apoptotic fraction was determined by PI staining and subsequent flow cytometry. Bars represent the mean percentages of apoptotic cells ± SD of at least three independent experiments. Significant changes compared to control are marked with an asterisk.
Fig 6.
CTX affects proliferation of spheroids in sensitive cell lines.
MTT assays were performed in 96-well plates with cells treated with EGFR stimulating or blocking reagents in monolayer (M) or forced suspension (FS) culture. Bars represent mean percentages ± SD of four independent experiments. Significant changes compared to control are marked with an asterisk.
Fig 7.
EGFR signalling regulates clonogenic survival of HNSCC cells derived from forced suspension cultures.
Cells were cultured as monolayer (M) or in forced suspension (FS) in the absence or presence of EGF, AREG or CTX. 72h later, cells were harvested, disaggregated to single cells and subjected to clonogenic survival analysis. Bars represent the surviving fractions (SF) ± SD from three independent experiments. Significant changes compared to control are marked with an asterisk.