Figure 4. NB tumor-derived adherent cells give rise to SMA+ perivascular stromal cells in vivo.

(A) Representative images of anti-EGFP immunohistochemistry in control and co-xenograft tumors. Tumors supplemented with primary adherent cells (lower panels) show EGFP positive cells (insets 1–4) with characteristic stromal cell morphology. Scale bars: 100 μm. (B) Immunofluorescent images detecting nuclei (blue), EGFP (green) and SMA (red) in two different co-xenografts. EGFP+ cells differentiate into SMA positive cells (yellow arrowheads) usually associated to other SMA+ cells (red arrowheads). Some of the double positive cells show characteristic perivascular localization (Co-XENO #132). Scale bars: 10 μm. (C) Representative immunofluorescent picture of a co-xenograft tumor detecting EGFP (green) and an endothelial cell-specific lectin (GSA I; red), illustrating the typical perivascular localization of primary adherent cells. Scale bar: 50 μm. (D) Immunohistochemistry against SMA in sections of control and co-xenograft tumors. Images were binarized (right panels) and the % of SMA positive area quantified. Scale bars: 100 μm. (E) Quantification of the % of SMA positive area in control and co-xenograft tumors (**p < 0.01, Mann-Whitney U-Test). (F, G) Tumor supplementation with NB5t derived stromal cells increase the expression of mesenchymal-like genes. (F) Quantitative PCR analysis of the expression of mesenchymal genes in control and co-xenograft tumors. Expression levels of ACTA2 (encoding for SMA), CD44, ENG, MEF2C, S100A10, TFGB1 and VIM mRNAs are shown normalized to their expression in control tumors (*p < 0.05, Student's t-test). (G) Correlation analysis of tumor weight (of both control and co-xenografts) versus mRNA expression levels of ACTA2, S100A10 and VIM (relative to GAPDH) (r = Pearson's correlation coefficient). These correlations indicate that the higher the expression of mesenchymal markers the bigger the tumor size.