Figure 6
Characterisation of the neoplastic phenotype. A) Frequency histograms of cellular DNA content in PD-neurospheres unexpanded and expanded for 41 pd. Lymphocytes were used as normal diploid internal standard. LAN-5 NB cells were used as aneuploid DNA content. Ploidy is expressed as DNA index (DI), which represents the ratio of relative G0/G1 DNA content of sample versus normal diploid lymphocyte. B) Papanicolau staining of PD-neurospheres unexpanded and expanded for 41 pd. LAN-5 NB cells were used as tumour control. The arrows indicate multinucleated abnormal cells with large prominent nucleoli. Original magnification: 40X. C) GD2 and N-Myc expression in secondary unexpanded PD-neurospheres (1 pd) and in PD-neurospheres expanded for different passages (3.6 pd and 41 pd). The negative sample was incubated with the corresponding isotype. The LAN-5 NB cells were used as a positive control. The cytograms shown are representative of three different experiments with similar results. D) Spontaneous malignant transformation of adherent PD-neurospheres expanded injected subcutaneously into the flanks of NOD.CB17-Prkdcscid/NCrHsd mice. Haematoxylin and eosin staining of tissue sections fixed in 4% formalin and paraffin embedding confirmed the infiltrative tumour phenotype. Original magnification: 40X. a) healthy muscle tissue; b and c) neoplastic nests of cells in muscle tissue. E) HLA-ABC immunohistochemistry reactivity of tissue sections fixed in 4% formalin and paraffin embedding confirmed the human origin. Original magnification: 40×. NB, neuroblastoma; PD, placenta-derived; pd, population doubling.