Abstract
Continuous passaging of cancer cell lines can drive phenotypic and genotypic divergence, potentially compromising the reliability of such models. In this study, we show that two late-passage strains (S1 and S2) of ovarian cancer cell line SKOV3, although authenticated via short tandem repeat (STR) profiling as identical, exhibit substantial differences in morphology, transcriptomic signatures, ability to form 3D cultures and chemotherapeutic responses. Notably, S1 formed compact 3D spheroids and exhibited enhanced epithelial-mesenchymal transition (EMT) pathway activity, whereas S2 displayed a more proliferative, MYC-driven phenotype with larger spheroid structures requiring higher seeding densities. Transcriptomic analysis revealed pathways associated with hypoxia, EMT and angiogenesis in 3D culture, highlighting the complexity introduced by dimensionality in tumour modelling. Critically, S1 showed higher sensitivity to doxorubicin than S2 (IC50 of 0.12 µM versus 1.28 µM, P=0.0001), indicating how clonal evolution can confound drug-response assays. Ultimately, our findings suggest that although STR profiling remains essential for cell line authentication, functionally distinct subpopulations can arise and coexist within the same culture, and their isolation may reveal divergent phenotypes that compromise reproducibility in preclinical cancer research.