Figure 2. Stage 4/M NB tumor-derived primary cultures contain a subpopulation of neural crest progenitor cells.

(A) Representative photomicrograph showing nuclei (DAPI; blue), Nestin (green) and GFAP (red) stainings in a NB tumor derived primary adherent culture. Nestin/GFAP double positive cells are pointed with yellow arrows. Inset: Expression of Sox2 (green) in NB5t primary adherent cells. Scale bar: 100 μm. (B) Representative picture showing the existence of GFAP/Nestin double positive cells (yellow arrows) in an original high-risk NB tumor tissue. Scale bar: 25 μm. (C) Primary cultures contain a subpopulation of cells that grow as spheres when cultured in non-adherent conditions. The bright field image on top shows typical spheres formed when NB tumor-derived adherent primary cells were cultured in low-binding conditions. Graph quantifies sphere-forming efficiency from 4 different tumor-derived samples, measured in primary, secondary and tertiary sphere passages, revealing the existence of a small but self-renewing fraction of sphere-forming progenitor cells. (D) Immunocytochemistry showing nuclei (DAPI; blue), Nestin (green) and GFAP (red) expression in cells from adherent cultures and from spheres grown in parallel. Nestin/GFAP double positive cells are pointed with yellow arrows. Scale bar: 100 μm. (E) Quantification of GFAP/Nestin double positive cells from 3 different primary cultures (NB5t, NB14t and NB27t) and their corresponding spheres. In general, spheres showed a clear increase in the percentage of double positive cells (from 5% to 31%) (**p < 0.01, Student's t-test). (F–H) Spheres are enriched in genes described as neural crest stem cell markers when compared to adherent cell cultures. (F) Quantitative PCRs showing a clear increment in the expression of Bmi1, Msi1 and Oct4 mRNAs (neural crest stem cell markers) in spheres when compared to their corresponding adherent cultures (level 1 line). The variability observed could be explained by tumor heterogeneity between patients. (G, H) Increase in mRNA expression was confirmed at protein level in primary cell cultures from tumor biopsy NB14t. (G) Immunohistochemistry showing the expression of Nestin (green) and Bmi1, Msi1 or Oct4 (red) in primary cell adherent cultures and spheres grown in parallel. Scale bars: 100 μm. (H) Quantification of expression levels based on the intensity of fluorescence (arbitrary units normalized to adherent) confirmed the increase in neural crest stem cell marker mRNA expression shown in (F) (*p < 0.05, Student's t-test). (I–L) Endothelin-1 (ET-1) increases survival and proliferation of neural crest progenitors within primary cultures. (I) Representative bright field images of spheres cultured in control conditions or after ET-1 treatment. (J) Top. Quantification of sphere-forming cell frequency illustrating how ET-1 increases the % of these cells in all primary cultures tested. Bottom. Quantification of sphere diameter showing how two of the three primary cell cultures also present a small but significant increase in sphere diameter when treated with ET-1. Both analyses together suggest an increase in survival and proliferation of neural crest progenitors treated with ET-1. (K, L) ET-1 mediated effect was confirmed by gene expression profiling of control and ET-1 treated spheres from three different primary cell cultures. (K) Hierarchical clustering and scatter plot obtained from a microarray gene expression analysis performed to compare control versus ET-1 treated spheres. (L) Ingenuity Pathway Analysis of genes differentially expressed (660 genes) after ET-1 treatment, predicting an increase in biofunctions like cell viability and cell survival, as well as in functions fully compatible with a neural crest origin of the spheres analyzed. (M) Immunofluorescent stainings of NB tumor-derived spheres after 4–5 days of differentiation in adherent conditions, with or without 15% serum. Cells were labeled with antibodies against nestin (stem cell marker), SMA (mesenchymal differentiation marker), S100b and GFAP (glial differentiation markers), and DDC and Tuj1 (neuronal differentiation markers). Scale bars: 100 μm.