Figure 3. NB tumor-derived adherent cells increase proliferation of NB cell lines in vitro and favor tumor growth in vivo.

(A) Diagram of the experimental design of in vitro co-cultures. IMR32 NB cells expressing a red fluorescent protein (IMR32-RFP) were cultured alone or with NB tumor-derived adherent cells at 1:1 ratio. After 4 days in culture, the % of IMR32-RFP proliferative cells (PHH3+) was measured. (B) Representative fluorescent images of cultures showing nuclei (blue), proliferative cells (PHH3+; green) and IMR32-RFP cells (red fluorescent protein+; red). Scale bars: 100 μm. (C) Quantification of the % of PHH3+ IMR32-RFP cells in control (IMR32-RFP cells cultured alone) and in co-cultures with adherent cells derived from 4 different NB tumors (NB5t, NB14t, NB21t and NB27t). Results are normalized to the % of PHH3+ cells in control cultures (*p < 0.05; **p < 0.01; ***p < 0.001, Student's t-test). (D) Quantification of IMR32-RFP proliferative cells (PHH3+) in co-cultures with NB5t cells, in the presence of AMD3100 and PAN-TGFβ inhibitors, always compared to control co-culture in the presence of vehicle (DMSO) (**p < 0.01; ***p < 0.001, Student's t-test). (E) Experimental design of co-xenografts. (F) Representative image of a mouse xenografted with IMR32 cells alone (control xenograft, left flank) or admixed with NB5t derived adherent cells (co-xenograft, right flank). (G) Examples of different tumors after 2 months of growth, illustrating the bigger sized phenotype of co-xenografts. Scale bars: 10 μm. (H) Quantification of tumor weight in control and co-xenograft tumors. (*p < 0.05, Mann-Withney U-Test). (I) Representative flow cytometry plots showing the incorporation of a small but consistent % of EGFP positive NB derived stromal cells in co-xenograft tumors. (J) Quantification of EGFP positive cells (%) in control and co-xenograft tumors (p = 0.057, Mann-Whitney U-Test).