Fig. 6: BMP signaling activity varies dramatically depending on neuroblastoma tumor site but is highly active in disseminated bone marrow metastases.
A Kaplan−Meier curves showing the survival of mouse. 5 mice were used in each group. Each mouse was implanted with luciferase-labeled CHP-134 cells (left panel) or BE(2)-C cells (right panel) by subcutaneous injection. RA, 13-cis retinoic acid; IRN, irinotecan; TMZ, temozolomide. The IRN + TMZ group is intended as a standard-chemo positive control. B GSEA plots summarizing relevant differentially expressed pathways in vehicle treated CHP-134 tumors in mouse versus untreated CHP-134 cells in vitro. Running Enrichment Scores are shown by colored lines. C Stripchart (left panel) showing ID1 expression (y-axis) in CHP-134 cells growing under different in vitro and in vivo conditions (x-axis). Heatmap (right panel) showing the expression of ID1-1D4 under the same conditions. D Western blot for phospho-SMAD1/5/9 in CHP-134 cells under various in vitro and in vivo RA treatment conditions. GAPDH was used as a loading control. The representative results of 3 independent replicates are shown. E Dot plot showing the top enriched pathways for differentially expressed genes in vehicle-treated CHP-134 mouse xenografts to vehicle treated CHP-134 cells in vitro. P-values (color) and odds ratios (x-axis) were calculated by hypergeometric test. Exact P-values are provided in the Source Data file. F Boxplots estimating BMP signaling activity based on the average TPM expression of ID1-ID3 (y-axis) in clusters of neuroblastoma cancer cells (dots), identified using acNMF from single-cell RNA-seq in different datasets (x-axis). P-values were calculated with two-sided Wilcoxon tests. G H scores (y-axis; see Methods) quantifying the IF staining intensity of phospho-SMAD1/5/9 in cells that also stain positive for PHOX2B (marking neuroblastoma cancer cells) in six different neuroblastoma patients (n = 6, each color indicates a patient). The x-axis shows the different conditions/sites. Note: the endothelial controls cells are PHOX2B negative. H Representative IF images from Patient #3 showing merged marker staining in bulk primary tumor (left upper panel), a lymphovascular invasion region of primary tumor (right upper panel), bulk metastatic tumor in the bone marrow (left lower panel) or disseminated cells in the bone marrow (right lower panel). White arrows and circles indicate representative cells positively stained with both PHOX2B and pSMAD1/5/9 (these cells appear yellow/orange, see scale bar, which is shown for illustrative purposes). Additional detailed images are included in Fig. S10A. I Scatter plot highlighting the overexpression of BMP ligands in ‘bone marrow stromal cells’ identified using single-cell RNA-seq on bone marrow aspirates from metastatic neuroblastoma patients. Dataset from Fetahu et al. J Expression of phosphorylated SMAD1/5/9 and c-PARP in CHP-134 and TGW. Cells were treated with DMSO or 0.03 µM ATRA in the presence of vehicle control or 10 ng/ml BMP recombinant proteins. CHP-134 was treated for 3 days and TGW was treated for 24 h. For CHP-134, all antibodies were incubated with the same membrane. GAPDH was used loading control. The representative results of 3 independent replicates are shown. K Viability of CHP-134 cells (left) treated with ATRA for 6 days in the presence of vehicle control or 10 ng/ml BMP recombinant proteins. Cell viability was measured with CellTiter-Glo. ATRA IC50 for CHP-134 (right) in the presence of each BMP was calculated with GraphPad Prism 9. Data represent the mean ± SD of 3 independent replicates. P values at the arrow indicated point for BMP1, 2, 4, and 7 are 0.37, 1.55 × 10−5, 7.42 × 10−6, and 2.83 × 10−5 (two-sided t-test). L Cell counting for CHP-134 treated as in (J). Dead cells were determined by trypan blue staining. Data represent the mean ± SD of 3 independent replicates. P values (DMSO vs ATRA) for vehicle, BMP1, 2, 4, and 7 are 1.6 × 10−3, 8.81 × 10−4, 3.58 × 10−5, 3.96 × 10−5, and 1.28 × 10−5 (two-sided t-test). Source data are provided as a Source Data file.
