Fig 8. Stromal cells enhance HOS-CSC migration via IL-6 and the expression of adhesion molecules.

(A) We assessed whether treatment with Tocilizumab affected HOS-CSC migration. MSC were treated with Tocilizumab [100 μg/mL] 2 hours prior CSC seeding. HOS spheres were trypsinized and single cells were let to migrate for 3 hours. As a control, medium only was added in the lower chambers, representative images; (B) Quantification of the migration assay shown in panel (A) (*p<0.05); (C) The expression levels of ICAM-1 were increased in HOS-CSC co-cultured with MSC. Data were obtained by Real Time PCR (*p<0.05) and confirmed by Western blot (D, representative image and densitometric quantification, T0 represents the protein expression level of parental cells from which CSC was obtained) (*p<0.05); (E) MSC were treated with 100 μg/mL Tocilizumab 24 hours prior CSC seeding. HOS-CSC spheres were then co-cultured by using tranwells with MSC and incubated for 6 hours. The RNA from CSC was then extracted and analyzed for the MET expression that shows a dramatic decrease in the absence of IL-6 (*p<0.05).