Abstract / Summary
Abstract Background Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype associated with rapid tumour growth, early metastatic dissemination and limited therapeutic options. Transcriptomic studies have provided extensive insight into TNBC biology, but how the mammary tissue transcriptome changes throughout tumour growth remains poorly understood. Methods We conducted stage-resolved RNA sequencing of mammary tissue collected at successive stages of orthotopic 4T1 tumour growth in immunocompetent BALB/c mice, alongside histopathological evaluation. Parental 4T1 cell line samples were included to differentiate transcriptional features associated with the in vivo tumour context. Results Histopathological evaluation confirmed progressive local invasion across successive stages, beginning with replacement of the cutaneous muscle layer and culminating in complete dermis replacement by the Late stage. Substantial transcriptomic variations from Control mammary tissue were already evident at the Early stages of tumour growth (G1-G2, days 3–7 post-inoculation). An individual analysis of the G1 stage, which lacked the macroscopically detectable features of a tumour, confirmed that the principal transcriptomic alterations were already present at this point. Across successive stages, the rate of gene-expression change was unevenly distributed, with the most pronounced shift occurring during the Medium 1 to Medium 2 transition, after which comparatively few additional changes were observed despite continued tumour growth. Functional enrichment analyses showed a progressive increase in the representation of extracellular matrix organisation and inflammatory pathways, alongside a decline in epithelial differentiation- and metabolism-related programmes. Comparison with the parental 4T1 cell line revealed that a substantial component of the in vivo transcriptomic landscape, particularly extracellular matrix and immune-related programmes, was not represented in cultured tumour cells, highlighting the contribution of the tissue microenvironment to tumour biology. Conclusions This stage-by-stage approach shows that extensive molecular alterations in mammary tissue are already observed within days of tumour cell inoculation, preceding the appearance of a macroscopic tumour mass. Transcriptomic change was not constant but centred on a pivotal transition between intermediate stages, after which most transcriptional programmes were already established. This study offers a stage-resolved molecular framework for interpreting tissue-level changes during TNBC progression, and highlights that a substantial part of this landscape reflects TME-associated programmes absent from cultured tumour cells alone.