Fig. 4.

IGF-1-dependent induction of cancer stem cell markers CD20, CD133, and CD271 (NGFR) on melanoma cells. a Melanoma cells (WM3749) co-cultured with TAB cells (days 3 and 9) show high expression of CD20 (middle and right panels) compared with the control culture (left panel) as determined by FACS analysis. Melanoma cells were co-stained with anti-CD146 (MCAM, PE-conjugated) and anti-CD20 (FITC-conjugated) antibodies to distinguish them from B cells, which are CD146-negative;percentages indicate co-expression of both markers on the malignant cells. b Melanoma cells (WM3749) co-cultured with TAB cells (day 6) show high expression of CD20, CD133 and CD271 (left panel) compared with minimal or low expression of those markers when tumor cells are co-cultured with NB cells (right panel). Co-culture of melanoma cells with TAB cells did not modulate the expression of CD144 (vascular-endothelial cadherin marker) that are normally expressed by aggressive melanomas (data not shown). Induction of CD20, CD133, and CD271 was blocked when anti-IGF-1 neutralizing antibody (10 μg/ml) was used in the co-culture (middle panel). Anti-IL-1, anti-PDGF or anti-VEGF antibodies had no effect on CD marker expression (data not shown). Percentages indicate co-expression of CD20, CD133, or CD271on CD146⁺ melanoma cells. Results are representative of two independent experiments. c Melanoma cells(WM3749, WM989 and 451Lu) cultured in the presence of recombinant IGF-1 (25 ng/ml) for 5 days showed high expression of CD20 or CD271 (red bars) compared with untreated cells (blue bars) as determined by FACS assay. Bar represents mean + SE of triplicate samples