Tumorigenicity of SKNAS iCSCs and monolayer cells in SCID/Beige mice. SKNAS iCSC and monolayer cells were injected s.c. into mice using different cell dosages (10⁶, 10⁴, and 10²). Tumor growth was monitored up to 109 d after cell injection. Each data point represented the average days that tumors took to reach 0.5 cm³ in size. Seven of seven mice injected with 10⁶ SKNAS iCSC and six of six mice injected with 10⁶ SKNAS monolayer cells developed tumors. Similarly, nine of nine mice injected with 10⁴ SKNAS iCSCs and six of six mice injected with 10⁴ SKNAS monolayer cells developed tumors. In contrast, nine of nine mice injected with 10² iCSCs developed tumors, whereas only seven of 10 mice injected with 10² monolayer cells had tumor growth.

Differential expression of CXCR4 in SKNAS iCSC and monolayer cell xenografts. (A) The expression of CXCR4 and MYC was examined by TaqMan qPCR in SKNAS monolayer cell and iCSC xenografts, as described in Fig. 1. SKNAS monolayer cells and the in vitro culture of SKNAS iCSCs (day 175) were included as controls. Expression levels of the genes were presented as fold change over SKNAS monolayer cells in SKNAS iCSCs at day 175, SKNAS monolayer cell xenografts, and SKNAS iCSC xenografts. (B) Immunohistochemical analysis showed that SKNAS iCSC xenografts were uniformly positive for CXCR4. In contrast, SKNAS monolayer cell xenografts were negative for CXCR4, with the exception of some rare cases in which a few cells were focally positive for CXCR4 staining.

Histopathological examinations of SKNAS monolayer cell and iCSC xenografts. The monolayer cell xenografts were composed of two distinct components having different cellular morphologies. Tumor cells in the first component were larger cells, and tumor cells in the other component were smaller in both cellular and nuclear size and had smaller nucleoli. These small tumor cells often produced neurites or neuropils (Arrows). The monolayer cell xenografts were thus classified as poorly differentiated NB. In contrast, iCSC xenografts were composed of uniformly large cells with vesicular nuclei and one or more prominent nucleoli, and thus were classified as totally undifferentiated LCNs.

Histopathological examinations of SKNAS iCSC xenografts and the human LCNs. H&E stained sections showed that the SKNAS iCSC xenografts resembled human undifferentiated LCNs histologically. The patterns of CXCR4 and MYC expression in SKNAS iCSC xenografts also resembled those in undifferentiated LCNs. Microscopic magnification of 400× was used for all pictures.

The adrenal gland metastasis of SKNBE(2)C iCSC clone 1 shows the LCN phenotype and expresses high levels of MYCN. The procedures used are described in Fig. 5.

MYC and MYCN expression in NB monolayer cell and iCSC xenografts. (A) In the SY5Y monolayer cell xenografts, the majority of MYC-expressing cells were intermingled with the minority of MYCN-expressing cells. In the SY5Y iCSC xenografts, MYC-negative cells were, in fact, MYCN-expressing cells, which formed small islands in the majority of MYC-expressing cells. (B and C) SKNBE(2)C and CHP134 were MYCN-amplified cells and uniformly expressed high levels of MYCN in both monolayer cell and iCSC xenografts. The procedures used are described in Fig. 5. Microscopic magnification of 400× was used for all pictures.