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. 2025 Aug 21;16:7689. doi: 10.1038/s41467-025-62673-2

Fig. 5. Drug screening on small round cell sarcoma tumoroids.

Fig. 5

A Schematic overview of the tumoroid drug screening workflow. Created in BioRender. Ringnalda, F. (2025) https://BioRender.com/mnd48e5. B Clustered heatmap of AUC values of each tumoroid model (x axis) and compound (y axis), showing AUC values after 120 h drug treatment in a dose-range of 0.1 nM to 10 µM. Low AUC (red) indicates high drug sensitivity while high AUC (blue) indicates low drug sensitivity. Highlighted clusters include (1) chemotherapy, proteases and histone deacetylase inhibitors, (2) mTOR inhibitors, (3) E3 ubiquitin-protein ligases and (4) MCL-1 inhibitors. C Clustered heatmap showing AUC values after chemotherapy treatment, compounds that are routinely used in ES and ELS patients marked in red. D Box plots showing the mean AUCs values of mTOR inhibitors in the indicated ES (n = 13, tumoroid lines), CDS (n = 6, tumoroid lines) and BRS (n = 2, tumoroid lines) tumoroids entities. The boxes represent the interquartile range, the line inside the box marks the median, and the whiskers extend to the lowest and highest values. Error bars represent SEM of individual indicated ES, CDS and BRS tumoroid enitities. P-values were calculated using a one-way Anova, Dunnet multiple comparisons test:*<0.05, **<0.005, ns not significant. Exact adjusted P-values: AZD8055, EWSR1::FLI1 vs. EWSR1::ERG/FEV = 0.0033, EWSR1::FLI1 vs. BCOR::CCNB3/KMT2D = 0.0157, EWSR1::FLI1 vs. CIC::DUX4 = 0.0035. Sirolimus, EWSR1::FLI1 vs. EWSR1::ERG/FEV = 0.0147, EWSR1::FLI1 vs. BCOR::CCNB3/KMT2D = ns, EWSR1::FLI1 vs. CIC::DUX4 = 0.0281. Temsirolimus, EWSR1::FLI1 vs. EWSR1::ERG/FEV = ns, EWSR1::FLI1 vs. BCOR::CCNB3/KMT2D = ns, EWSR1::FLI1 vs. CIC::DUX4 = ns. E Box plots showing the mean AUCs values of P53 pathway activators in the indicated ES, CDS and BRS tumoroids entities. The boxes represent the interquartile range, the line inside the box marks the median, and the whiskers extend to the lowest and highest values. Error bars represent SEM of individual indicated TP53 wildtype (p53WT) (n = 14) vs TP53 mutant (p53MUT) (n = 7) tumoroids lines. P-values were calculated using a two-tailed Student’s t test: ****<0.0001. Exact P-values: Idasanutlin ≤ 0.0001, Milademetan ≤ 0.0001, Siramadlin ≤ 0.0001. F Clustered heatmap of compounds targeting the apoptosis pathway, CDS tumoroids showing specific sensitivity towards MCL-1 inhibitors (in red). G Validation screen with MCL-1 inhibitors, dose-response curves of S63845, MIK665 and AZD5991 for indicated ES and CDS tumoroid cultures. Error bars represent SD, each consisting of triplicate measurements. SEM standard error of mean, SD standard deviation, ES Ewing sarcoma, CDS CIC::DUX4 sarcoma, BRS BCOR-rearranged sarcoma. Source data are provided as a Source Data file.