Fig 2: Low ST8SIA1 expression correlates with low surface GD2 and mesenchymal features.a, Schematic showing the complete ganglioside synthesis pathway. Enzymes responsible for conversion of each ganglioside are labeled in bold and branch points are colored. b, Parental SH-EP (top) or CHLA-255 (bottom) cell lines were sorted based on GD2 expression into GD2low (red) or GD2high (black) isogenic cell lines, respectively. c, qPCR analysis comparing expression for ST8SIA1, B4GALNT1 and ST3GAL5 in the SH-EP-GD2high and SH-EP-GD2low (top) or CHLA-255-GD2high and CHLA-255-GD2low (bottom) isogenic cell line pairs. Data derived from a single experiment with 4 technical replicates, experiment was completed once. d, Cell viability for Kelly-GD2low and NB-SD with or without GD3 synthase overexpression and co-cultured with NK cells at an E:T ratio of 1:2 and in the presence of absence of 1 µg/mL dinutuximab for 8 h (n = 4 samples per treatment group). Data are shown as mean ± s.d. Significance determined by one-way ANOVA and Tukey’s post-hoc test. e, Flow cytometry plot showing GD2 expression in Kelly-GD2low, SK-N-AS and NB-SD cell lines with or without constitutive expression of ST3GAL5. f, qPCR analysis measuring ST8SIA1 expression in parental or SK-N-BE(2)-NOTCH3IC cells. Data derived from a single experiment with 4 technical replicates, experiment was repeated twice. ns = not significant. Representative data from flow cytometry were confirmed in two independent experiments.

Fig 3: GD3S downregulation is correlated with AMT.a,b, Pearson correlations were generated for ST8SIA1 against expression for all other genes in neuroblastoma cell lines found in the CCLE (n = 31) and rank-ordered based on most to least correlated. GSEA was performed on neuroblastoma-specific, adrenergic (A) or mesenchymal (B) gene sets. q-values are shown. c, Box-and-whisker plot comparing log2(TPM + 1) normalized expression for ST8SIA1 and B4GALNT1 in ADRN (n = 25) or MES (n = 6) cell lines found in the CCLE. Boxes show median, 25% quartile and 75% quartile; whiskers denote smallest and largest values within 1.5 times the interquartile range. Significance was determined by two-tailed Student’s t-test for each individual gene. d, qPCR analysis for ST8SIA1 in three models of AMT (SK-N-BE(2)-PRRX1, KP-N-YN-PRRX1 and SH-EP/SH-SY5Y). Data are derived from a single experiment with four (SK-N-BE(2) and KP-N-YN) or three (SH-EP/SH-SY5Y) technical replicates, and the experiment was repeated twice. e, Western blot showing protein expression for GD3S and GD2S in three AMT models. GAPDH is included as a loading control. Note that GAPDH for SH-EP and SH-SY-5Y are the same bands as shown in Extended Data Fig. 1e. f, Flow cytometry showing GD2 expression in parental, PRRX1-transduced or PRRX1-transduced + GD3S SK-N-BE(2) and KP-N-YN cell lines. g, Cell viability for SK-N-BE(2) and KP-N-YN cell lines shown in panel f. Cell lines were co-cultured with NK cells at an E/T ratio of 1:2 for 48 h and in the presence or absence of 1 μg ml−1 dinutuximab (n = 3 samples per treatment group). Data are shown mean ± s.d. Significance determined within each cell line by one-way ANOVA and Tukey’s post-hoc test. Representative data from flow cytometry and western blots were confirmed in two independent experiments.

Fig 4: GD2 downregulation is correlated with reduced GD3S expression.a, Scatterplot showing the correlation of the percentage of GD2+ cells in each cell line against the expression of GD3 synthase (ST8SIA1) from the CCLE. Linear regression line is shown, and gray cloud denotes 95% two-tailed confidence interval. R2 and P values are indicated. TPM, transcripts per million. b, Schematic showing the b-series ganglioside synthesis pathway. Gene names for enzymes that are responsible for the conversion of each product are labeled in italics, and protein abbreviations are labeled in bold. c, Scatterplot showing the correlation of the percentage of GD2+ cells against the expression of GD2 synthase (B4GALNT1) from the CCLE. Linear regression line is shown, and gray cloud denotes 95% two-tailed confidence interval. R2 and P values are indicated. d, Box-and-whisker plots comparing log2(TPM + 1) normalized expression for selected ganglioside synthase enzymes in GD2-low (n = 7) or GD2-high (n = 7) cell lines found in the CCLE data. Boxes show median, 25% quartile and 75% quartile; whiskers denote smallest and largest values within 1.5 times the interquartile range. Dots outside of the whiskers denote significant outliers. Significance was determined by two-tailed Student’s t-test within each gene. e,f, qPCR analysis comparing expression of ST8SIA1 (e) or B4GALNT1 (f) in representative GD2-high (n = 9 biological replicates) or GD2-low (n = 7 biological replicates) cell lines. Significance was determined by two-tailed Student’s t-test within each gene. Data are shown as mean ± s.d. g, qPCR analysis comparing expression for a panel of ganglioside synthase enzymes in Kelly-GD2high and Kelly-GD2low cells. Data derived from a single experiment with four technical replicates, experiment was repeated twice. h, Representative flow cytometry plots for GD2 expression in Kelly-GD2low, SK-N-AS or NB-SD cell lines transduced to overexpress GD2 synthase, GD3 synthase or both together. i, Cell viability for SK-N-AS cells ±GD3S overexpression that were co-cultured with NK cells at an E/T ratio of 1:2 for 48 h in the presence or absence of 1 μg ml−1 dinutuximab (n = 3 samples per treatment group). Data are shown mean ± s.d. Significance determined by one-way ANOVA and Tukey’s post-hoc test. Representative data from flow cytometry were confirmed in two independent experiments.