Figure 5. NELL2^highCD133^highEWS-FLI1^high and NELL2^lowCD133^lowEWS-FLI1^low populations in Ewing sarcoma display phenotypes consistent with high and low NELL2 signaling, respectively.

(A) NELL2 expression is heterogeneous in Ewing sarcoma tumor. NELL2 expression in surgically resected Ewing sarcoma tumor was examined by immunohistochemistry (brown signals). Scale bar: 60 μm.

(B) NELL2 expression is heterogeneous in Ewing sarcoma cells and correlates with EWS-FLI1 expression. NELL2 and EWS-FLI1 expression in A-673 cells was assessed by anti-NELL2 (red) and anti-FLI1 C terminus (green) immunofluorescent staining. The nuclei were stained with DAPI. Scale bar: 10 μm.

(C) NELL2 expression correlates with CD133 expression in Ewing sarcoma cells. NELL2 and CD133 expression in A-673 cells was assessed by anti-NELL2 (green) and anti-CD133 (AC133, red) immunofluorescent staining. The nuclei were stained with DAPI. Scale bar: 10 μm.

(D) The CD133^low population displays lower NELL2, EWS-FLI1, BRG1, BAF250A, and BAF155 levels than the CD133^high population. A-673 cells were incubated with anti-CD133 (AC133) antibody and were sorted into the CD133^high and CD133^low populations. The expression of indicated proteins was assessed by immunoblotting.

(E) The CD133^low population displays reduced EWS-FLI1 target gene expression. The RNA expression of indicated genes was assessed by qRT-PCR and is presented after normalization to the levels in the CD133^high population (blue). The expression of EWS-FLI1 target genes is reduced in the CD133^low population (red) (n = 3).

(F) The CD133^low population displays slower growth than the CD133^high population.

(G) The CD133^low population displays reduced NELL2 and EWS-FLI1 expression. A-673, CHLA-9, EW8, TC71, TC32, and SK-N-MC Ewing sarcoma cells were sorted into the CD133^high and the CD133^low populations, and the RNA expression of CD133, NELL2, and EWS-FLI1 was assessed by qRT-PCR (n = 3).

(H) The CD133^low population displays increased filopodia. Actin cytoskeleton was visualized by phalloidin staining (red). Cell nuclei were stained by DAPI (blue). Scale bars: 10 μm. The quantification of filopodia is shown in Figure S4D.

(I) NELL2 silencing inhibits the proliferation of both CD133^high and CD133^low populations.

(J) The CD133^low population displays reduced sphere formation; *p < 0.05 (3 independent experiments).

(K) The CD133^low population displays reduced xenograft tumorigenicity (n = 5, p < 0.05).

(L) The CD133^low population displays reduced migration; *p < 0.05 (3 independent experiments).

(M) Prolonged cisplatin or doxorubicin treatment enriches the CD133^low population.