Multiple Myeloma predominantly grows in bone, causing extensive bone destruction. Despite targeted therapies, relapse is common and the disease remains incurable. To develop new treatments we need a better understanding of myeloma cell engraftment, dormancy and growth in the skeleton. We hypothesize that myeloma cells engage in a specific bone niche in which they can reside dormant, resist chemotherapy and be activated to contribute to disease relapse. To address this, we developed intravital imaging to visualize dormant and activated myeloma cells in mice.
5TGM1eGFP murine myeloma cells were labeled with a membrane dye (DiD). In vitro, DiD label was lost as cells divided, suggesting this label distinguishes dormant (DiDhigh,+ve) from proliferating cells (DiDNeg). DiD labelled 5TGM1eGFP cells were injected into C57BLKalwRij mice and treated with melphalan (3 times/week, 5mg/kg) or vehicle, from day 14-28. Using intravital microscopy, individual dormant DiD+ve/GFP+ve cells were localized opposed to bone surfaces at 7, 14, 21, or 28 days post injection. At days 14 and 21, discrete activated DiDNeg/GFP+ve colonies were visualized, expanding to day 28 and localized distant from bone surfaces. In contrast, dormant DiD+ve/GFP+ve cells remained close to bone surfaces. Dormant DiD+ve/GFP+ve tumour cells were present after melphalan treatment, whereas growing tumour cells were reduced (>97%). Further, following recovery from melphalan treatment, DiD+ve/GFP+ve cells reduced. Microarray analysis of DiDHigh/GFP+ve cells demonstrated a transcript profile consistent with cell cycle arrest and chemo-resistance.
Taken together this shows that intra-vital microscopy can identify dormant myeloma cells and follow their activation and growth over time. Furthermore, we show that dormant cells are retained following chemotherapy and are available to repopulate the tumor. This longitudinal intravital imaging technique is applicable to a broad range of skeletal tumors and may provide insights into the fate of dormant cells, mechanisms behind drug resistance and disease relapse.