In order to treat metastatic

In order to treat metastatic bone disease effectively all steps involved in the metastatic cascade need to be taken into account as for example the interactions between primary and secondary sites, tumour cell–bone cell cross talk and osteomimicri [8]. Therefore, concomitant targeting of both the tumour and the bone microenvironment is likely to be required. Bisphosphonates have been combined with chemotherapy agents in models of bone metastasis from both breast and prostate cancer (reviewed in [2,9]). An intensive dosing regimens of docetaxel and risedronate (4mg/kg docetaxel twice a week, 150μg/kg risedronate 5 times a week), has been shown to eliminate osteolytic bone disease and cause a substantial inhibition of tumour growth in a MDA-231luc model, compared to the single agents [10]. Similarly, Brubaker et al. found that repeated administration of zoledronic clofibrate and docetaxel (100μg/kg zoledronic acid twice a week and 20mg/kg docetaxel every 2 weeks) was superior to the single agents at inhibiting growth of established LuCaP 23.1 prostate cancer bone metastases [11]. Using clinically achievable dosing regimens we have previously demonstrated a substantial, sequence-dependent, anti-tumour activity of doxorubicin (2mg/kg) and zoledronic acid (100μg/kg) in vivo. Six cycles of weekly administration of the two agents induced significant and sustained reduction in the growth of s.c. MDA-MB-436 xenografts [12,13], osseous breast tumours [14] and inhibition of development of spontaneous mouse mammary tumours [15]. The clinical relevance of these studies is unclear, as patients currently receive zoledronic acid only once every 3–4 weeks, compared to the 6 weekly doses, and agents are routinely given on the same day rather than after a 24-hour gap. In a model of advanced bone metastasis with established lytic lesions, we have shown that a single administration of zoledronic acid given 24h after the first cycle of weekly doxorubicin does reduce intra-osseous tumour growth [16]. However, starting treatment at this late stage of the disease did not have major impact on progression or survival, suggesting that earlier interventions are required to achieve significantly improved outcomes. In the present study we have therefore investigated whether administration of a single round of clinically achievable combination therapy at an early stage of bone metastasis development can prevent tumour progression in bone. Due to the increasing importance of the microenvironment in osseous tumour growth [17], we have also performed a detailed analysis of the effects of the therapeutic agents on bone cells, both in the presence and absence of tumour. Our data show that a single dose of zoledronic acid modifies both osteoblasts and osteoclasts, regardless of the presence of tumours. Despite zoledronic acid causing sustained increase in trabecular bone volume, there was no reduction in bone tumour burden without the addition of doxorubicin. Early administration of combination therapy caused a substantial but transient suppression of tumour growth, indicating that repeated courses of treatment are required for optimal anti-tumour effects.
Materials and methods
Results This study used an established model of bone metastasis where intracardiac injection of MDA-MB-231 breast cancer cells result in tumour growth in the long bones of nude mice. As shown in Fig. 1, treatment was administered on days 2–3 following tumour cell injection, to allow tumour cell homing and initial colonisation of skeletal sites. Animals were sacrificed either on day 15 or day 23. A parallel experiment was performed using animals without tumours, terminated on day 15, to allow assessment of treatment-induced effects in a tumour-free bone microenvironment.
Discussion Our data show that, despite zoledronic acid having a substantial effect on bone, tumour growth was only reduced in animals that received combination therapy. This is in agreement with a number of studies reporting a lack of anti-tumour effects after bisphosphonate monotherapy. The basis of combination therapy is to effectively reduce tumour growth by targeting both the tumour and the microenvironment at the metastatic site. In agreement with previous studies [12–14,16], we found that only tumours from the combination group exhibited significant differences in apoptosis on day 15, mirroring the reduction in tumour area. No changes in active tumour cell proliferation were detected at either time point. In addition, genes regulating apoptosis and proliferation showed to be significantly altered following 6 weekly cycles of combination therapy [14] were unchanged after the single cycle given in the present study. This is probably due to differences in treatment scheduling and sample collection points, as in the earlier investigations tumours were collected for analysis 24h after the final administration of repeated cycles of combination therapy. The data suggest that apoptotic cell death and reduction in proliferation is likely to be an acute effect of combination therapy, and therefore no longer detectable 2–3 weeks following administration of treatment. These results clearly demonstrate that repeated cycles of combination therapy is required to maintain an anti-tumour in bone effect beyond 1–2 weeks.