Chemotherapy resistance and stemness in mitotically quiescent human breast cancer cells identified by fluorescent dye retention

Metastatic recurrence in breast cancer is a major cause of mortality and often occurs many years after removal of the primary tumour. This process is driven by the reactivation of disseminated tumour cells that are characterised by mitotic quiescence and chemotherapeutic resistance. The ability to reliably isolate and characterise this cancer cell population is critical to enable development of novel therapeutic strategies for prevention of breast cancer recurrence. Here we describe the identification and characterisation of a sub-population of slow-cycling tumour cells in the MCF-7 and MDA-MB-231 human breast cancer cell lines based on their ability to retain the lipophilic fluorescent dye Vybrant^® DiD for up to six passages in culture. Vybrant^® DiD-retaining (DiD+) cells displayed significantly increased aldehyde dehydrogenase activity and exhibited significantly reduced sensitivity to chemotherapeutic agents compared to their rapidly dividing, Vybrant^® DiD-negative (DiD-) counterparts. In addition, DiD+ cells were exclusively capable of initiating population re-growth following withdrawal of chemotherapy. The DiD+ population displayed only partial overlap with the CD44⁺CD24^-/low cell surface protein marker signature widely used to identify breast cancer stem cells, but was enriched for CD44⁺CD24⁺ cells. Real-time qPCR profiling revealed differential expression of epithelial-to-mesenchymal transition and stemness genes between DiD+ and DiD- populations. This is the first demonstration that both MCF-7 and MDA-MB-231 human breast cancer lines contain a latent therapy-resistant population of slow-cycling cells capable of initiating population regrowth post-chemotherapy. Our data support that label-retaining cells can serve as a model for identification of molecular mechanisms driving tumour cell quiescence and de novo chemoresistance and that further characterisation of this prospective tumour-reinitiating population could yield novel therapeutic targets for elimination of the cells responsible for breast cancer recurrence.