Characteristics and transcriptional regulators of spontaneous epithelial–mesenchymal transition in real-world genetically unperturbed non-spindle breast carcinoma

Background Although epithelial–mesenchymal transition (EMT) has been well-characterized in experimental models, convincing histopathological evidence of EMT in primary carcinomas has only been obtained for rare spindle carcinomas. The existence, characteristics, and transcriptional regulators of spontaneous EMT in real-world, genetically unperturbed non-spindle carcinoma remain underexplored. Methods We used primary culture combined with RNA sequencing (RNA-seq), single-cell RNA-seq (scRNA-seq), and in situ RNA-seq to explore the characteristics and transcription factors (TFs) associated with potential spontaneous EMT in non-spindle breast carcinoma. Results Our primary culture revealed carcinoma cells expressing diverse epithelial–mesenchymal traits, consistent with epithelial–mesenchymal plasticity. Importantly, carcinoma cells undergoing spontaneous EMT did not necessarily exhibit spindle morphology, even when undergoing complete EMT. EMT was a favored process, whereas mesenchymal–epithelial transition appeared to be crucial for secondary tumor growth. Through scRNA-seq, we identified TFs that were sequentially and significantly upregulated as carcinoma cells progressed through the EMT process, which correlated with increasing VIM expression. Once upregulated, the TFs remained active throughout the EMT process. ZEB1 was a key initiator and sustainer of EMT, as indicated by its earliest significant upregulation in the EMT process, its exact correlation with VIM expression, and the reversal of EMT and downregulation of EMT-upregulated TFs upon ZEB1 knockdown. The correlation between ZEB1 and vimentin expression in triple-negative breast cancer and metaplastic breast carcinoma tumor cohorts further highlighted its role. The immediate upregulation of ZEB2 following that of ZEB1 suggested their functional cooperation in EMT. This finding, together with that of a lack of correlation of SNAI1 , SNAI2 , and TWIST1 expression with the mesenchymal phenotype, indicated EMT-TFs have a context-dependent role in EMT. Upregulation of EMT-related gene signatures during EMT correlated with poor patient outcomes, highlighting the biological importance of the model. Elevated EMT gene signatures and increased ZEB1 and ZEB2 expression in vimentin-positive compared to vimentin-negative carcinoma cells within the corresponding primary tumor tissue confirmed ZEB1 and ZEB2 as intrinsic, instead of microenvironmentally-induced, EMT regulators, and vimentin as an in vivo indicator of EMT. Conclusions Our findings provide insights into the characteristics and transcriptional regulators of spontaneous EMT in non-spindle carcinoma in real-world scenarios.