Most bisphosphonate (BP) drugs, such as zoledronic acid (ZOL), inhibit bone resorption by blocking the synthesis of isoprenoid lipids essential for protein prenylation in osteoclasts. This post-translational modification with a lipid tag is essential for the membrane targeting and function of small GTPases such as Rho and Rac, that regulate the cytoskeletal dynamics underlying cell morphology, polarisation, adhesion and migration. Contrary to the dogma that BPs only affect osteoclasts, we have recently shown that systemic BP treatment can inhibit protein prenylation in vivo in certain cells outside the skeleton, for example in peritoneal macrophages in mice. To better understand how BPs may affect macrophage function, we used live cell imaging and image analysis tools to quantify changes in the morphology, adhesion, polarisation and migration of the IC-21 peritoneal macrophage cell line.

Treatment of IC-21 cells with 1-10µM ZOL for 48 hours caused a concentration-dependent inhibition of protein prenylation (based on the detection of unprenylated Rap1A by western blotting and unprenylated Rab GTPases using an in vitro prenylation assay). 10µM ZOL reduced cell adhesion by approximately 50%, without affecting cell viability. Treated macrophages also failed to polarise and did not form clear lamellipodia (leading end) or a uropod (lagging end). Cell motility (speed and displacement) was impaired by >60%. In a matrix degradation assay to measure MMP activity associated with podosomal attachments, ZOL treatment appeared to enhance localised matrix degradation, probably due to decreased cell migration. These defects in cell morphology, polarisation and migration were proportional to the degree of inhibition of protein prenylation, and addition of isoprenoid lipid completely restored prenylation, cell morphology and cell migration.

These imaging approaches allow the prediction of how macrophage behaviour is affected by BPs such as ZOL and will facilitate the ex vivo analysis of cells obtained from mice or humans following BP treatment.