Nitrogen-containing bisphosphonates have previously been shown to inhibit Ewing sarcoma (ES) tumour cell growth and to promote apoptosis of ES cells, possibly by upregulating osteoprotegerin expression; they also inhibit primary ES tumour growth in a mouse model of ES. ES cells have been shown to express RANKL. In this study we investigated the effect of the bisphosphonate zoledronic acid (ZA) and the anti-RANKL inhibitor osteoprotegerin on the viability of ES cells in vitro

ES cell lines TC71 and SK-N-MC and primary EWS-Fli1 positive cells derived from a case of ES were cultured and treated with ZA (200nM to 200uM), osteoprotegerin (1ng/ml to 1ug/ml) or vehicle control. After 3 days, viability was assessed using PrestoBlue  reagent and results normalised to cells treated with vehicle control.

ZA inhibited ES Cell viability with an IC50 of approximately 10uM in the established cell lines TC71 and SK-N-MC and with an IC50 of 20uM in low-passage primary ES tumour cells.  Osteoprotegerin did not decrease the viability of cells at any concentration tested.

Although ZA inhibited ES cell viability, the IC50 of ZA was achieved at very high concentrations which were unlikely to be achieved clinically; it is possible that with the known strong binding of bisphosphonate to bone, high concentrations of ZA might be achieved in the bone microenvironment at a lower dose. However, in this case it would not be “free” but bound to bone and interaction with ES cells may not be predictable. It is of interest that when the sensitivity of osteosarcoma cell lines to ZA is similarly measured in vitro, a similar IC50 to the one we obtained is observed. Our results also suggest that the effect of anti-RANKL agents will be on osteolysis associated with ES tumour growth rather than on the viability of ES tumour cells.