Abstract
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.
Highlights
Metastatic recurrence in advanced breast cancer is a major cause of patient morbidity and mortality
We demonstrate the novel application of Vybrant® DiD for identification, isolation and characterisation of a latent, slow-cycling, label-retaining cell population in the oestrogen receptor-positive MCF-7 and triplenegative MDA-MB-231 human breast cancer cell lines
The effects of DiD staining on the proliferation of MCF-7 and MDA-MB-231 cell lines was assessed by haemocytometric counting of viable cell number in unstained and DiD-stained cultures at 24-h intervals; no significant differences were detected at any time point in either cell line, confirming that the cell labelling protocol had no adverse effects on cell viability and growth (Supplementary Fig. 3c)
Summary
Metastatic recurrence in advanced breast cancer is a major cause of patient morbidity and mortality. Yumoto et al [6] described the identification of slowly cycling sub-populations in a number of human prostate cancer cell lines based on their persistent retention of fluorescent lipophilic dye. Expanding on these findings, Wang et al [7] demonstrated that label-retaining prostate cancer cells differentially up-regulate expression of various haematopoietic stem cell niche-associated markers and were significantly more metastatic in vivo when compared to the rapidly dividing cell population isolated from the same parental culture. The link between these slow-cycling, label-retaining populations and chemoresistance has remained largely unstudied and it remains unclear whether label-retaining cells represent a tumour recurrence-initiating population
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
