Figure 4.

Enhanced self-renewal capacity in brain metastasis (BM) cells upon mast cell (MC) stimulation. BM cells were subjected to tumorsphere formation assay in order to examine the self-renewal stem-like cell population in the BM cells and the influence of MC mediators after coculture. (A) Tumorsphere formation assay of patient-derived BM cells U3333MET and the BM cell line NCI-H1915 (upper panel). Sphere forming capacity of the cells was compared before and after coculture with MCs by counting the number of formed spheres. Results are presented as number of spheres formed per 4,000 seeded cells (lower panel). (B) Expression analysis of stem cell markers in the BM cells before and after coculture with MCs. β-actin expression level was used for normalization. For each gene, the expression level in the control cells was considered as 1 and the expression levels after coculture was calculated relative to it. (C) In-cell ELISA to evaluate SOX2 and CD133 expression in BM cells before and after coculture with MCs. All the experiments were performed three times in triplicates and mean values + SEM was plotted, ns, not significant; **p < 0.01, ***p < 0.001.