Fig. 5. Chemotherapy induces a transition toward MuSC-like states in aRMS.

(A) Association between cluster-associated signatures and aRMS patient outcome. Violin plots show log fold change distributions between deceased and living patients assuming no association (10⁷ simulation replicates). Black dots represent the calculated associations. (B) Experimental workflow. (C) Number of dedifferentiating drug hits at 1 μM. (D) Shared dedifferentiating drugs at 1 μM ranked on the basis of the average dedifferentiating score. A score of zero represents the baseline score of untreated controls. (E) Immunofluorescence quantification of aRMS-1 cells exposed to 10 nM vincristine or 1 μM etoposide for 72 hours; ordinary two-way ANOVA with uncorrected Fisher’s LSD. (F) qRT-PCR data generated with aRMS-1 cells exposed to 10 nM vincristine sulfate or 50 μM 4-HC for 48 hours; ordinary two-way ANOVA with uncorrected Fisher’s LSD. (G) WB images of aRMS-1 cells exposed to vincristine or 4-HC for 48 hours. GAPDH was used as loading control; samples were loaded on the same gel. (H) Representative FACS plots of aRMS-1 cells treated with 1 nM vincristine or 10 μM 4-HC for 48 hours. (I) Experimental workflow. (J) qRT-PCR data generated with aRMS-1 tumors collected after in vivo treatment with vincristine (10 mg/kg); ordinary two-way ANOVA with uncorrected Fisher’s LSD. (K) WB images of aRMS-1 PDX tumors treated in vivo with vincristine. Samples were loaded on the same gel. (L) Expression of myogenin and Ki-67 as determined by immunohistochemistry in aRMS-1 PDX tumors following in vivo treatment with vincristine or vehicle. (M) Proposed model of treatment selection following chemotherapy in aRMS. *P < 0.05; **P < 0.01; ***P < 0.001; ****P ≤ 0.0001. Data are represented as means ± SEM of the indicated number of biological replicates. Untr., untreated; Vin, vincristine sulfate; 4-HC, 4-hydroperoxycyclophosphamide; Veh, vehicle; FC, fold change.