Nature Genetics: doi: /ng Supplementary Figure 1. Phenotypic characterization of MES- and ADRN-type cells.
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1 Supplementary Figure 1 Phenotypic characterization of MES- and ADRN-type cells. (a) Bright-field images showing cellular morphology of MES-type (691-MES, 700-MES, 717-MES) and ADRN-type (691-ADRN, 700- ADRN, 717-ADRN) cell lines. Note attached growth of MES-type lines, while ADRN-type cell lines grow as semiattached spheres. Scale bar, 100 m. (b) PROM1 (encoding the CD133 antigen) mrna expression in the three MES and ADRN cell line pairs shown in a. (c) FACS analysis of CD133 expression on the isogenic cell lines 691-MES and 691-ADRN. (d) Box plots showing frequency of CD133 + cells in three MES and ADRN cell line pairs as measured by FACS. Each circle represents an individual FACS analysis. (e) Box plots showing motility of MES or ADRN cells as measured in Transwell migration assays. Each circle represents the average number of migrated cells from quadruplicate experiments. (f) Unsupervised cluster analysis (Spearman rank correlation) of the 1,000 genes with the highest standard deviation in the four isogenic pairs of MES and ADRN cell lines. Four MES cell lines cluster on the left, and four ADRN cell lines cluster on the right. The same results were obtained for the 500 or 1,500 genes with the highest standard deviation. (g) k-means clustering for the 1,000 genes with the highest standard deviation in the series of 33 neuroblastoma cell lines mapped in Figure 1c. The 500 or 1,500 genes with the highest standard deviation gave similar results.
2 Supplementary Figure 2 Spontaneous in vitro interconversion and in vivo tumorigenicity of MES and ADRN cells. (a) Frequency of CD133 + cells in clones grown from FACS-sorted CD133 AMC700B cells. (b) Survival of mice xenografted with FACSsorted CD133 + or CD133 cells of cell line AMC700B. Mice were sacrificed when tumors were up to 1 cm in diameter. (c) IHC analysis of the tumors grown from xenografted CD133 + or CD B cells. Tumors were stained for dopamine -hydroxylase (DBH) and vimentin (VIM). HE, hematoxylin and eosin staining.
3 Supplementary Figure 3 Examples of H3K27ac profiles of MES- and ADRN-specific super-enhancer-associated genes. (a) Five MES-specific genes with associated super-enhancers (SEs). (b) Box plots showing expression of the genes shown in a in the MES and ADRN counterparts of the four isogenic cell line pairs. (c) Five ADRN-specific genes associated with super-enhancers. (d) Box plots showing expression of the genes shown in c in the MES and ADRN counterparts of the four isogenic cell line pairs. Whiskers in b and d denote the interval within 1.5 times the interquartile range (IQR) of the median.
4 Supplementary Figure 4 mrna expression levels and super-enhancer status of all TFs in pairs of MES and ADRN cell lines of isogenic origin. (a d) XY plots showing the expression of all TFs. TFs with MES-specific expression identified in the MES-specific signature are indicated in orange. TFs with ADRN-specific expression identified in the ADRN-specific signature are indicated in blue. TFs of the signature associated with super-enhancers are indicated by a bold colored dot. (a) 691-MES and 691-ADRN cells. (b) 717-MES and 717-ADRN cells. (c) 700-MES and 700-ADRN cells. (d) SH-EP2 and SH-SY5Y cells.
5 Supplementary Figure 5 GATA3 binds to the epicenters of super-enhancers of ADRN-specific TFs. (a) The most prominent GATA3 binding peak 19 within super-enhancers of ADRN super-enhancer-associated TFs was used to center a ±1-Mb region. Data are from cell line SH-SY5Y. Gene bodies within the region are indicated in thin stripes, and signature TFs are indicated with thick stripes. The blue dot indicates GATA3, zoomed in c. (b) The same regions are shown, now showing the H3K27ac signal 19. (c) Example of the GATA3 and H3K27ac profiles in the region of the GATA3 gene and its associated super-enhancers. The zoomed region shows that GATA3 peaks binds to the epicenter of the H3K27ac domains.
6 Supplementary Figure 6 H3K27ac profiles of nine cell lines for PHOX2A and PHOX2B. (a) PHOX2A. (b) PHOX2B. Horizontal blue bars indicate super-enhancer (SE) regions.
7 Supplementary Figure 7 PRRX1 induces adrenergic-to-mesenchymal reprogramming. (a) Protein blot analysis of neuroblastoma cell lines 691-ADRN, SH-SY5Y and SK-N-BE(2C) with doxycycline-inducible expression of PRRX1 (t = 96 h). Immunoblots were analyzed for PRRX1 induction, mesenchymal marker SNAI2 and adrenergic markers PHOX2B and DBH. -actin is used as a loading control. Dox, doxycycline. (b) PRRX1 induces the migratory potential of 691-ADRN, SH-SY5Y and SK-N-BE(2C) cells. Each circle represents the number of migrated cells per high-power field. Four high-power fields per Transwell were analyzed. (c) Time-course mrna analysis of SK-N-BE(2C) cells with inducible PRRX1 expression. Downregulation kinetics between 8 and 196 h after PRRX1 induction were clustered by k-means analysis in four groups (clusters 1 4). Below each cluster are representative examples of four genes. mrna levels as measured by Affymetrix array of PRRX1-expressing cells are indicated in red, and control cells (without Dox) are shown in blue. The x axis depicts time in hours; the y axis depicts expression values for indicated genes. (d) Western blot analysis of cell line SK-N-AS treated by CRISPR Cas9 with control grna (84, 86) or PRRX1-gRNA (clones 1, 2a). Lane 2 is underloaded. (e) Western blot analysis of cell line GI-MEN treated by CRISPR Cas9 with control grna (84, 86) or PRRX1-gRNA (clones 1, 2a, 2b). (f) Western blot analysis of PRRX1 in 691-MES cells expressing IPTG-inducible control shrna or PRRX1 shrna (646, 647, 648). -actin is shown as a loading control. (g) Analysis of the ADRN and MES scores of the cell lines with silencing of PRRX1 shown in d f. Inactivation of PRRX1 does not result in significant shifts. See Supplementary Data for uncropped images of western blots used in a and d f.
8 Supplementary Figure 8 Relationship between the MES and ADRN signature scores and molecular and clinical variables and the prognostic value of PRRX1 in a series of 122 primary neuroblastoma tumors. (a) HE staining of tumor N712T, which is a highly mesenchymal neuroblastoma as indicated in b, shows ~90% tumor cell content. (b h) Mesenchymal and adrenergic signature scores in primary neuroblastoma tumors in relation to percentage tumor cells in sections used for mrna profiling (b), amplification of MYCN (c), LOH1p (d), loss of chromosome 11 (e), INSS stage (f), survival (g) and age (h). Box plots show the median signature score for each group. Whiskers denote the interval within 1.5 times the interquartile range (IQR) of the median. Tumors for which parameters were not determined are not shown in b h. Cell line pairs are numbered 1 4 with MES and ADRN counterparts shown in orange and blue, respectively. (i l) Kaplan Meier analysis of the relationship between PRRX1 expression and overall survival in a series of 122 neuroblastoma tumors 12 (i,j) and a neuroblastoma series analyzed by RNA seq 22 (k,l). Patients were classified in two groups with PRRX1 expression above or below the median value. The analysis was performed for all patients in the series (i,k) or for stage 3 and 4 patients only (j,l).
9 Supplementary Figure 9 PRRX1 is expressed in neuroblastoma cells. (a) Representative false-color image of double-ihc for MYCN (blue) and PRRX1 (red). MYCN + PRRX1 + cells are shown in yellow. Scale bar, 100 m. (b) Percentage of MYCN PRRX1 double-positive cells relative to all PRRX1 + cells in six stage 4 neuroblastoma tumors. (c) Dot plot visualization of MAML3 mrna expression levels (Affymetrix probeset _at) in three independent neuroblastoma series (red) as compared to eight other tumor types (blue) and nine normal tissues (green). (d) Hematoxylin and eosin staining (HE; left) and double-ihc for MAML3 (blue, nuclear) and the ADRN markers DBH (middle; red, cytoplasmic) and TH (right; red, cytoplasmic) on serial neuroblastoma sections from a representative stage 4 neuroblastoma. Str, Schwannian stroma; Nbl, neuroblasts with small round blue morphology. Scale bar, 100 m.
10 Supplementary Figure 10 Mesenchymal neuroblastoma cells are chemo-resistant in vitro and are enriched in post-therapy tumors in vivo. (a) Box plots showing sensitivity to cisplatin, doxorubicin or etoposide of pairs of MES- and ADRN-type cells of isogenic origin. MTT assays were used to determine viability after cisplatin, doxorubicin or etoposide treatment. MES- and ADRN-type cells in orange and blue, respectively. Whiskers denote the interval within 1.5 times the interquartile range (IQR) of the median. (b) IHC analysis of the mesenchymal marker PRRX1 in tumor biopsies taken before and after chemotherapy treatment (patients 1 and 2) or after treatment (patients 3 and 4). Scale bar, 50 m.
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