Figure 1. Characterization of stromal phenotype in primary adherent cell cultures isolated from stage 4/M NB tumor biopsies.

(A) Schematic view of cell isolation protocol from tumor biopsies. (B) Representative bright field images of primary adherent cells isolated from two different NB biopsies (NB5t and NB14t) showing the characteristic stromal-like phenotype of cultured cells. (C) Inmunofluorescent labeling of mesoderm-derived fibroblasts with TE-7 marker, or neural crest-derived myofibroblasts with smooth muscle actin (SMA) in a control human fibroblast cell line (hFIB) and in three different NB tumor-derived cell cultures (NB5t, NB14t and NB27t). (D-G) Primary cells expressed mesenchymal-like but not neuroblastic markers. (D) Gene expression analysis comparing a NB primary cell culture (blue bars) and its original tumor sample (orange bars) (NB5t). Genes were selected according to their relationship to mesectodermal or neuroblastic phenotypes and their reported enrichment in neural crest stem cells (see text for references). Based on this result, a panel of surface markers was selected to extend the gene expression analysis to 5 NB primary cell cultures and 3 NB commercial cell lines with a reported neuroblastic phenotype. (E) Representative flow cytometry plots showing expression of MSCA1, CD44 (mesenchymal markers), CD56 and CD57 (neuroblastic markers) in NB primary adherent cells from NB5t tumor and in the neuroblastic cell line IMR32. (F) Analysis of flow cytometry data showing the expression level (% of positive cells) for each surface marker tested in each NB tumor derived primary cell culture or in NB commercial cell lines. (G) Quantification of data shown in (F) (*p < 0.05, Mann-Whitney U-test), comparing primary cells versus cell lines. Scale bars in (B) and (C): 100 μm.