Bisphosphonate (BP) drugs rapidly target the skeleton and are the gold standard treatment to inhibit bone destruction in patients with osteoporosis and metastatic bone disease. However, BPs also have anti-cancer effects outside the skeleton; in some preclinical models they reduce primary tumour growth, and adjuvant BP therapy increases survival of patients with breast cancer and multiple myeloma. The exact mechanisms underlying these anti-cancer effects are unknown since BPs are considered to only affect bone-resident osteoclasts in vivo. To answer this question, we determined the cell types capable of internalising fluorescently-labelled BP in 4T1 mammary tumours in vivo. Within minutes of tail vein injection, intravital 2-photon imaging revealed the diffusion of BP into tumour tissue from the leaky, disorganised tumour vasculature. BP then appeared to bind to small, granular microcalcifications within the tumour tissue. Intravital imaging revealed that tumour-associated macrophages (TAMs) rapidly internalised BP by pinocytosis and by engulfing these BP-coated microcalcifications. Flow cytometric analysis of the tumours 24hr later confirmed that uptake occurred predominantly in TAMs and not tumour epithelial cells.
We also identified a patient with breast cancer in which the BP 99mTc-MDP (used for SPECT/CT bone scintigraphy) localised to the primary mammary carcinoma. Histological analysis of the resected tumour post-surgery revealed the presence of granular microcalcifications similar in appearance to those in the mouse 4T1 tumours, and some of which were closely associated with CD68+ TAMs.
These studies provide conclusive evidence that BPs can be rapidly internalised by macrophages outside the skeleton. The leaky vasculature of tumours facilitates the local diffusion of BP, where it binds to microcalcifications within the tumour that are engulfed by TAMs. Given the important role of TAMs in promoting tumour progression and metastasis, our studies suggest that the anti-tumour activity of BPs in cancer patients occurs indirectly via effects on these cells.