Cell-to-cell variability in PAX3:FOXO1 expression determines tumorigenic potential in rhabdomyosarcoma

The rhabdomyosarcoma (RMS) cell pool is phenotypically and functionally heterogeneous. Low passage cell lines established from Myf6Cre,Pax3:Fkhr,p53 mouse RMS and primary human RMS cultures were used to demonstrate marked heterogeneity in PAX3:FOXO1 (P3F) expression at the single-cell level. In mouse Myf6Cre,Pax3:Fkhr,p53 RMS cells, expression of P3F is directed by the Pax3 promoter and coupled to an eYFP fluorescent marker. YFPlow/ P3Flow mouse RMS cells included 87±0.2% G0/ G1 cells, differentially expressed transcripts involved in extracellular matrix interaction and reorganized their actin cytoskeleton to produce a cellular phenotype characterized by more efficient adhesion and migration compared to YFPhigh/ P3Fhigh cells. By contrast, 49±3.2% of YFPhigh/ P3Fhigh cells were in the G2/ M phase of the cell cycle, and higher P3F expression correlated with higher proliferation rates. These differences translated into higher tumor-propagating cell frequencies in limiting dilution analyses and higher clonal activity of YFPlow/ P3Flow compared to YFPhigh/ P3Fhigh cells. Both YFPlow/ P3Flow and YFPhigh/P3Fhigh cells gave rise to mixed clones, consistent with fluctuations in P3F expression at the cellular level over time. Finally, exposure to the anti-tropomyosin compound TR100 disrupted the actin cytoskeleton and reversed more efficient migration and adhesion in YFPlow/ P3Flow RMS cells. We speculate that conversion from one phenotype to another may result in adaptive plasticity and may provide a critical advantage during tumor progression Overall design: We performed ATAC-Seq on YFP high / P3F high and YFP low / P3F low cells isolated from mouse rhabdomyosarcoma cells U23674.