Figure 2. Human NB Formation in Mouse-Human Chimeras.

(A) Schematic representation of the experiment. E8.5 mouse embryos injected with hNCCs and monitored for tumor induction.

(B and C) Bioluminescent imaging shows human tumor formation at the age of 4 months (B) and macroscopic tumor growth at the age of 6 month (C).

(D) Tumors of chimeric mice dissected at P120 (left; DF, dark filed) express tdTomato (middle) and eGFP (right), indicating their origin from donor hNCCs.

(E) eGFP expression within tumors was confirmed by IHC (scale bar indicates 10 μm).

(F and G) The human tumors were found to express MYCN and ALK by IF (left; scale bar indicates 100 μm) and IHC staining (right; scale bar indicates 10 μm).

(H and I) CHNBs express the typical cancer hallmarks Ki67 (H) and γH2AX (I); scale bar indicates 20 μm.

(J) H&E and IHC show that CHNB express the typical NB markers synaptophysin (SYP), nestin (NES), tyrosine hydroxylase (TH), and chromogranin A (CgA) similarto NB patient samples. Dox-treated hNCCs, subcutaneously injected into immunocompromised mice, formed xenograft tumors (left column), which did not express these NB markers. See IHC quantifications in Figures S4F–S4I; scale bars indicate 100 μm.

(K) Western blot analysis of different NB models show expression of MYCN and ALK along with ALK (pALK Tyr¹⁵⁰⁷) self-activation in CHNBs, in PDX NBs (NB-patient-derived xenografts) with amplified MYCN (MYCN^amp), with and without mutation in ALK (ALK^F1174L or ALK^WT), and in mouse NB tumors of GEMM overexpressing MYCN and ALK^F1174L (Th-MYCN+ALK^F1174L). Activation of MAPK and PI3K/AKT pathways in CHNBs and other MYCN+ALK^F1174L NB models was shown by phosphorylation of pERK1/2 Thr²⁰² Tyr²⁰⁴, pAKT Ser⁴⁷³, and pAKT Thr³⁰⁸. Total ERK1/2, AKT, and GAPDH are presented for controls. 1× = 1 volume of loading; 2× = twice the loading volume.

(L) IHC staining for pERK1/2 and pS6K, confirming MAPK and PI3K/AKT activity in CHNB tumors (scale bar indicates 10 μm).