Skeletal metastasis is a hallmark of advanced prostate cancer (PCa), with no effective treatments currently available. Rare, dormant PCa cells, residing in ‘niches’ in the skeleton are believed to initiate development of bone metastasis and contribute to disease relapse. Progress in targeting bone metastasis is limited due to challenges with identifying and studying dormant cells. The aim of the present study was to develop an approach to identify dormant PCa cells in the skeleton.
PC3-GFP human PCa cells were labelled with a fluorescent membrane-bound dye (vybrant-DiD®). This is lost as the dye is shared between daughter cells (DiD-) and retained in non-dividing or dormant cells (DiD+). DiD-labelled cells were monitored in vitro for loss of label using fluorescent microscopy and flow cytometry. Labelled cells were seeded onto the endocranial side of excised calvaria from neonatal mice, cultured ex vivo and imaged by two-photon microscopy. For in vivo studies, DiD-labelled cells were injected into the left ventricle of the heart of 6-week-old male BALB/c-nude mice and bones isolated after 6 weeks for ex-vivo analysis.
Fluorescent microscopy and flow cytometric analysis revealed a progressive loss of DiD label in PC3-GFP cells over 14 days in vitro, suggesting all cells proliferated. In contrast, when PC3-GFP cells were cultured on calvaria they retained DiD label and PC3-GFP-DiD+ cells were still present 21 days post-seeding even in the presence of proliferating cells. In vivo, label-retaining, dormant PC3 cells were detectable at 6-weeks post-injection in the long bones. Two-photon imaging demonstrated the presence of dormant PC3-GFP-DiD+ cells opposed to bone surfaces in intact femurs even in the presence of a growing tumour (PC3-GFP-DiD-).
These studies show that dormant PCa cells can persist in the bone microenvironment, even in the presence of proliferating cells, suggesting only limited numbers of dormant cells are activated to form overt metastasis.