Breast cancer commonly metastasises to the skeleton where it is difficult to treat. Breast cancer cells are believed to reside in the skeleton in a dormant state for prolonged periods. Dormant cancer cells (DCCs) are thought to play a pivotal role in driving tumour growth in bone and are likely responsible for disease recurrence following chemotherapy. Currently, the molecular pathways regulating cancer cell dormancy remain to be determined. DCC’s have very low abundance in bone and are therefore difficult to detect and study. The aim of this study was to develop an approach to identifying and studying rare dormant breast cancer cells in bone.
MDA-MB-231LUC2 or MDA-MB-231GFP cells were labelled with a fluorescent membrane dye (DiD). In-vitro, DiD-labelling was progressively lost as cells proliferated, suggesting that DiD-labelling distinguishes dormant cells (DiDhi) from proliferating cells (DiDneg). In-vivo, flow cytometry of bone marrow (BM) isolated from mice injected with DiD-labelled MDA-MB-231cells via the intracardiac route, demonstrated a population of proliferating DiDneg cells from day 14, increasing in number to day 21. In addition, DiDhi dormant cells were detected from day 7 and were present in BM throughout the time-course. The number of DiDhi cells ranged from 0-26 cells per bone. Two-photon imaging of explanted tibiae demonstrated the presence of dormant, DiD retaining cells opposed to the endosteal bone surface. A limited number of DiDneg proliferating tumour colonies was also identified. DiDhi cells were found even in the presence of actively growing tumours suggesting that not all dormant cells are activated.
Using this novel technique, we have identified a distinct population of rare and long-lived cancer cells in a murine model of breast cancer colonisation to bone. These findings will facilitate advances in understanding dormant cancer cell biology which is crucial for disease prevention, progression and, importantly, recurrence.