Characterisation of Stemness in Soft Tissue Sarcoma using Functional and Omics-based Approaches

Soft tissue sarcomas (STS) comprise a rare and highly heterogeneous group of mesenchymal tumours encompassing more than 80 histological subtypes. Research has largely focused on recurrence, metastasis and chemoresistance, with the cancer stem cell (CSC) model proposed as a potential explanation for aggressive tumour behaviour. However, evidence for CSCs in STS remains inconclusive and appears subtype dependent. The aim of this study was to characterise stemness and its functional relevance in Sheffield-derived STS cell lines through integrated functional and omics-based approaches. Previous assessment of putative CSC markers revealed distinct, subtype-specific expression patterns. Functional assays, including invasion assay and autophagy assay, alone were insufficient to reliably define stemness. While genomic and transcriptomic profiling did not conclusively confirm discrete CSC populations, PI3K-Akt, MAPK, RAP1 and Ras pathways, which are associated with stemness, exhibited elevated activity. Nutritional depletion assay identified stress-resistant subpopulations capable of re-entering proliferation and colony formation in cell lines with cellular viability after nutritional stress higher than 0.1%. Survival following metabolic stress correlated with increased cellular dormancy and enhanced spheroid formation. Transcriptomic and genomic analyses post-nutritional depletion indicated upregulation of pro-survival and stemness-associated networks, activation of PI3K-Akt and MAPK signalling, suppression of cell cycle and OXPHOS pathways, and increased chromosomal instability. Collectively, these findings demonstrate that stemness in STS is dynamic, context- and subtype-dependent state, highlighting the necessity of integrating functional validation with omics profiling to inform subtype-specific therapeutic strategies.