GD2 and lineage marker expression in TH-MYCN tumors, primary explants and cell lines, and human neuroblastoma cell lines. (a) TH-MYCN+/+ neuroblastomas (NB), previously established and propagated TH-MYCN+/+ and human NB cell lines were examined for surface GD2 expression by flow cytometry in single, live, CD45- cells, and NCAM1+ and CD56+ for mouse and human NBs, respectively. Cell lines were passaged for 3 weeks to assess GD2 stability. (b) Fresh TH-MYCN+/+ tumors were explanted and carried in tissue culture to monitor the GD2+ proportion of tumor cells over time ex vivo. (c) Immunoblot detection of a principal adrenergic marker, Phox2b, principal mesenchymal marker, Yap1, and β-tubulin loading control. IMR5 and SHEP are neuroblastoma cell lines with a predominantly adrenergic or mesenchymal lineage, respectively. TH-MYCN+/+ primary tumors are predominantly adrenergic marker expressing. TH-MYCN+/+ tumor-derived cell lines are largely mesenchymal marker expressing when propagated ex vivo (>3 months, 3000-series cell lines) whereas marker expression is heterogeneous during earlier passage ex vivo (5000-series cell lines). (d) Immunocytochemistry and immunohistochemistry to detect Phox2b and Yap1 from FFPE cell line pellets or primary tumors are shown, concordant with immunoblot results.