A single administration of combination therapy inhibits breast tumour progression in bone and modifies both osteoblasts and osteoclasts

Abstract

We have previously shown that repeated sequential administration of doxorubicin, followed 24 h later by zoledronic acid, inhibits tumour growth in models of established breast cancer bone metastasis. As breast cancer patients only receive zoledronic acid every 3-4 weeks, the aim of the current study was to establish the anti-tumour and bone effects of a single administration of doxorubicin/zoledronic acid combination therapy in a bone metastasis model. MDA-MB-231-GFP cells were injected i.c. in 6-week-old nude mice. On day 2, animals received PBS, doxorubicin (2 mg/kg i.v.), zoledronic acid (100 μg/kg s.c.) or doxorubicin followed 24 h later by zoledronic acid. Anti-tumour effects were assessed on days 15/23 by quantification of apoptotic and proliferating cells and changes in expression of genes implicated in apoptosis, proliferation and bone turnover. Bone effects were assessed by μCT analysis, bone histomorphometry and measurement of serum markers. A tumour-free control group was included. Combination treatment reduced bone tumour burden compared to single agent or PBS control and increased levels of tumour cell apoptosis on day 15, but this was no longer detectable on day 23. Animals receiving zoledronic acid had increased bone density, without evidence of tumour-induced lesions. Bone histomorphometry showed that zoledronic acid caused a decrease in osteoblast and osteoclast numbers and an increase in osteoclast size, in both tumour-free and tumour-bearing animals. Our data show that although zoledronic acid modifies the bone microenvironment through effects on both osteoblasts and osteoclasts, this does not result in a significant anti-tumour effect in the absence of doxorubicin.

Keywords: Bisphosphonate; Bone metastasis; Breast cancer; Combination therapy; ELISA, Enzyme-linked immunoassay; GFP, Green fluorescent protein; H&E, Haematoxylin and eosin; NBP, Nitrogen-containing bisphosphonate; Ob, Osteoblast; Oc, Osteoclast; PINP, Procollagen type I N-terminal propeptide; TRAP, Tartrate-resistant acid phosphatase; μCT, Microcomputed tomography.

Fig. 1

Study protocol assessing treatment-effects on tumour-free and tumour-bearing bones. For the tumour study, balb/c nude mice (female, 6 weeks) were injected with MDA-MB-231-GFP cells on day 0. All animals (including tumour-free mice) were treated on day 2 with PBS, doxorubicin (2 mg/kg i.v.), zoledronic acid (100 μg/kg s.c.) or doxorubicin followed 24 h later with zoledronic acid, animals were sacrificed on day 15 or 23.

Fig. 2

Effects of combination therapy on tumour burden. (A) Analysis of total tumour area (mm²) performed on a minimum of 2 non-serial sections per sample using a 4x objective. One way ANOVA and Tuckey post test, * is p<0.05, ** is p<0.01, *** is p<0.001, ns=not significant. (B) Example Goldner's trichrome stained section depicting extra- and intra-osseous tumour foci. (C) Number of mice on day 23 with intra- and extra-osseous tumour colony detected by histology.

Fig. 3

Effects on caspase-3 and Ki67/BrdU. Histological sections were stained for caspase-3 to detect apoptotic tumour cells. Data are expressed as number of positive cells per tumour area in mm² for (A) day 15 and (B) day 23. Staining for (C) BrdU (day 15) or (D) Ki67 (day 23) was performed to detect tumour cell proliferation At least 2 non-serial sections per sample were analysed using a 10x objective. One way ANOVA and Tuckey post test, ** is p<0.01 vs. PBS control, ns=not significant.

Fig. 4

Effects on bone structure and integrity in the absence of tumour. Quantification of trabecular bone volume per tissue volume (BV/TV) for tumour-free animals sacrificed on day 15. One way ANOVA and Tuckey's post test, *** is p<0.001 compared to control and +++ is p<0.001 compared to doxorubicin.

Fig. 5

Effects on bone structure and integrity in tumour-bearing bones. Analysis of osteolytic bone disease was carried out for the proximal tibia and the distal femur in animals sacrificed on (A) day 15 and (B) 23. Data are expressed as trabecular bone volume per tissue volume (% BV/TV). One way ANOVA and Tuckey's post test, *** is p<0.001 compared to naïve, PBS or dox; ^ is p<0.05 vs. naïve and ∼ is p<0.05 vs. zol; ns=not significant. Cross sections taken from μCT scans of the proximal tibia and images of GFP foci in bone are shown in for (C) day 15 and (D) day 23.

Fig. 6

Treatment effects on osteoblast and osteoclast numbers in tumour-free bone. Animals were treated with PBS, 2 mg/kg dox, 100 μg/kg zol or combination therapy on day 2. Osteoblast (A) and osteoclast (B) numbers were analysed per mm trabecular bone surface on a minimum of 2 non-serial TRAP stained sections per sample. One way ANOVA and Tuckey post test, * is p<0.05 and *** is p<0.001 compared to PBS control; + is p<0.05 vs. dox; ns=not significant.

Fig. 7

Treatment effects on osteoblast and osteoclast numbers in tumour bearing bone. Animals were treated with PBS, 2 mg/kg dox, 100 μg/kg zol or combination therapy on day 2. Osteoblast (A) and osteoclast (B) numbers were analysed per mm trabecular bone surface on a minimum of 2 non-serial TRAP stained sections per sample. One way ANOVA and Tuckey post test, * is p<0.05, ** is p<0.01 and *** is p<0.001 compared to PBS control; + is vs. dox and ^ is vs. naïve; ns=not significant.

Fig. 8

Effects on osteoclast size. Measurement of osteoclast size (expressed as mean area in mm²) for osteoclasts in (A) tumour-free mice and (B) tumour-bearing animals. Data are shown as mean±SEM, One way ANOVA and Tuckey post test, * is p<0.05, ** is p<0.01, *** is p<0.001, ns=not significant. Black columns: grey columns: day 23. (C) Example images of TRAP stained osteoclasts (red) for each treatment group on day 15, scale bar =50μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)