Fig. 1: Fusion-driven pediatric sarcoma cell line strains exhibit exceptional genomic, transcriptomic, and phenotypic stability compared to adult carcinoma strains.

a Newly purchased A-673 EwS, HeLa cervix carcinoma and MCF-7 breast carcinoma wild type cell lines (A-673_1, HeLa_1 and MCF-7_1) were kept in culture for six months (m6; A-673_2, HeLa_2 and MCF-7_2) and twelve months (m12; A-673_3, HeLa_3 and MCF-7_3). In addition, multiple strains for each cell line were collected from seven, three, and two laboratories, respectively, and arbitrarily numbered as A-673_4 to A-673_9, HeLa_4 to HeLa_5, and MCF-7_4 to MCF-7_5. Single-cell clones derived from A-673 cell lines with a neutral manipulation (*, A-673_10) or an inducible shRNA construct targeting its EWSR1::FLI1 translocation (**, A-673_11) were included. ATCC, American Type Culture Collection, DSMZ (German Collection of Microorganism and Cell Cultures). b Relative in-exon SNVs counts in cancer genes for A-673, HeLa, and MCF-7 after six months of continuous culture, using the respective initial time point (m0) values as reference (m6 vs. m0). c Heatmap comparing the status (homozygous for reference allele, alternate allele, or heterozygous) of non-synonymous SNPs in 11 A-673 strains. The left color bar depicts chromosomes; the right color bar depicts different SNP-IDs (N = 1,599). d Transcriptomic PCA of 11 A-673, five HeLa, and five MCF-7 cell line strains (N = 10,256 transcripts). e Circle plot depicting coefficient of variation (CV) of expressed genes per chromosome (top 60% quantile) for all A-673, HeLa, and MCF-7 cell line strains. f Volcano plot of DEG resulting from comparing the two A-673, HeLa, and MCF-7 strains with the highest variance (A-673_7 vs A-673_3, HeLa_5 vs HeLa_3 and MCF-7_5 vs MCF-7_3). The red dots denote significantly differentially expressed genes (BH adjusted P < 0.01; |FC | > 1). g Combined transcriptomic PCA of our A-673 and HeLa datasets and that of Liu et al. (HeLa_Liu) (N = 13,569 transcripts). h Relative gene expression of A-673, HeLa, and MCF-7 cell lines after long-term culture for twelve months (m12 vs m0). Outer violin curves denote the kernel density. Boxplots display the interquartile range and the mean, two-sided Wilcoxon signed-rank test. i Left, collective variability in drug response of all A-673, HeLa, and MCF-7 cell line strains depicted as the standard error of ED, each compound is shown as a black circle, one-sided Wilcoxon signed-rank test. Right, standard error of ED for each specific screened compound. j Spearman’s ρ of drug response across 11 A-673, five HeLa, and five MCF-7 cell line strains. The dotted black line shows the median (one-sided Wilcoxon rank-sum test). k Raw viability of A-673, HeLa, and MCF-7 cell lines subjected to each compound (1 µM) included in the drug screening after 0, 6, and 12 months of continuous long-term culture (m0, m6, and m12).