Zoledronic acid has differential antitumor activity in the pre- and postmenopausal bone microenvironment in vivo

Abstract

Purpose: Clinical trials in early breast cancer have suggested that benefits of adjuvant bone-targeted treatments are restricted to women with established menopause. We developed models that mimic pre- and postmenopausal status to investigate effects of altered bone turnover on growth of disseminated breast tumor cells. Here, we report a differential antitumor effect of zoledronic acid (ZOL) in these two settings.

Experimental design: Twleve-week-old female Balb/c-nude mice with disseminated MDA-MB-231 breast tumor cells in bone underwent sham operation or ovariectomy (OVX), mimicking the pre- and postmenopausal bone microenvironment, respectively. To determine the effects of bone-targeted therapy, sham/OVX animals received saline or 100 μg/kg ZOL weekly. Tumor growth was assessed by in vivo imaging and effects on bone by real-time PCR, micro-CT, histomorphometry, and measurements of bone markers. Disseminated tumor cells were detected by two-photon microscopy.

Results: OVX increased bone resorption and induced growth of disseminated tumor cells in bone. Tumors were detected in 83% of animals following OVX (postmenopausal model) compared with 17% following sham operation (premenopausal model). OVX had no effect on tumors outside of bone. OVX-induced tumor growth was completely prevented by ZOL, despite the presence of disseminated tumor cells. ZOL did not affect tumor growth in bone in the sham-operated animals. ZOL increased bone volume in both groups.

Conclusions: This is the first demonstration that tumor growth is driven by osteoclast-mediated mechanisms in models that mimic post- but not premenopausal bone, providing a biologic rationale for the differential antitumor effects of ZOL reported in these settings. Clin Cancer Res; 20(11); 2922-32. ©2014 AACR.

Figure 1. Effects of ovariectomy on bone structure and bone turnover

(a) Bone volume 0, 1, 2, 3, 4, 5 and 8 weeks following ovariectomy. (b) Photomicrographs of Goldners’ stained histological sections of the tibia and reconstructed μCT images at baseline and 8 weeks following ovariectomy or sham operation. Osteoclast activity was analysed by measuring serum levels of TRACP 5b and osteoblast activity assessed by measuring P1NP (c) in ovariectomised mice compared with sham operated animals. Numbers of osteoclasts and osteoblasts (d) lining the bone surface 0, 1, 2, 3, 4, 5 and 8 weeks following ovariectomy. Data shown are mean ± SEM and * represents a p value of < 0.05.

Figure 2. Ovariectomy increases of the number of MDA-MB-231 breast cancer cell colonies in bone

Experimental outline (a) and (b) photographs of luciferase expressing MDA-MB-231 cells inoculated 7 days after ovariectomy or sham operation and 56 days following tumour cell inoculation. The percentage of mice with detectable tumours and mean tumour volume ± SEM shown as numbers of photons per second per tumour (c). Bone volume following ovariectomy in mice injected with MDA-MB-231 cells (mean ± SEM) and uCT images representing bone architecture (d). All data are shown for 56 days following tumour cell inoculation and * represents a p value of < 0.05.

Figure 3. Ovariectomy stimulates growth of established breast cancer cells in long bones of 12-week-old immunocompromised mice

Experimental outline (a) and photographs of luciferase expressing MDA-MB-231 cells inoculated 7 days before ovariectomy or sham operation growing in mice 49 days following tumour cell inoculation (b). Percentage of mice with detectable tumours and mean tumour volume ± SEM is shown as numbers of photons per second per tumour (c). Effects on bone volume following ovariectomy in mice injected with MDA-MB-231 cells (mean ± SEM) and μCT images representing bone architecture (d). All data are shown for 49 days following tumour cell inoculation and * represents a p value of < 0.05.

Figure 4. Zoledronic acid inhibits bone resorption and reduces tumour take in ovariectomised mice

(a) Experimental outline. (b) Histogram showing mean ± SEM % of mice with detectable bone tumours in control and zoledronic acid treated ovariectomised and sham operated mice 35 days following tumour cell inoculation. (c) Bone volume compared with trabecular volume 28 days following ovariectomy and 31 days following zoledronic acid treatment in mice pre-injected with MDA-MB-231 cells (mean ± SEM). (d) μCT images representing bone architecture at the end of the experimental protocol. * represents a p value of < 0.05 compared with sham control and ** a p value of < 0.05 compared with sham control and OVX control.

Figure 5. Non-proliferating tumour cells are present in proximal trabecular bone of mice without detectable metastasis

Confocal images of disseminated DiD labelled tumour cells (red cells highlighted with yellow block arrows) that have homed to bone but not formed tumours in tibiae of ovariectomised control mice (a), ovariectomised zoledronic acid treated mice (b), sham control (c) and sham zoledronic acid treated animals (d).