Fig. 4

Multicellular tumour spheroids assay determining the effect of EGF and S1PR2 (A) MCTSs of H357, H400 and H413 cell lines after hanging drop culture for 1 day. Spheroids consisted of approximately 500 cells. The size and circularity of the spheroids on the embedding day were similar. (B) Images of spheroids embedded in 3 mg/ml collagen after incubation with treatments for 48 h. C-E) EGF at 20 ng/ml resulted in an increase in both spheroid fragmentation (note the number of clusters) and the distance of invasion as well as a decrease in circularity of the main cluster. The effects of EGF on these characteristics of invasion decreased following inhibition of S1PR2. These morphological parameters did not change after treatment following the addition of the S1PR2 agonist following EGF treatment. (Scale bar shown represented 100 μm; three biological replicates were performed with N = 10 for each experiment; ANOVA followed by post-hoc Tukey tests, * = p < 0.05, ** = p < 0.01, data presented as mean ± 1 SD.)