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Location matters: osteoblast and osteoclast distribution is modified by the presence and proximity to breast cancer cells in vivo

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Abstract

Bone metastasis is a common incurable complication of breast cancer affecting around 70% of patients with advanced disease. In order to improve outcomes for these patients, the cellular and molecular mechanisms underlying bone metastasis need to be established. The majority of studies to date have focused on end-stage disease and little is known about the events taking place following initial tumour cell colonisation of bone. Here we report the results of a longitudinal study that provides detailed analysis of the spatial and temporal relationship between bone and cancer cells during progression of bone metastasis. Tumour growth in bone was initiated by intra-cardiac inoculation of MDA-MB-231-GFP breast cancer cells in immunocompromised mice. Differentiating between areas of bone in direct contact with the tumour and areas distal to the cancer cells but within the tumour bearing bone, we performed comprehensive analyses of the number and distribution of osteoclasts and osteoblasts. Tumour colonies were detectable in bone from day 10, while reduced trabecular bone volume was apparent from day 19 onwards. Cancer-induced changes in osteoblast and osteoclast numbers differed substantially depending on whether or not the cells were in direct contact with the tumour. Compared to naïve controls, areas of bone in direct contact with the tumour had significantly reduced osteoblast but increased osteoclast numbers, whereas the reverse was found in distal areas. Our data demonstrate that tumour cells induce substantial changes in the bone microenvironment prior to the appearance of bone lesions, suggesting that early therapeutic intervention may be required to oppose the tumour-induced changes to the microenvironment und thus tumour progression.

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Abbreviations

Oc:

Osteoclast

Ob:

Osteoblast

PINP:

Procollagen type I N-terminal propeptide

uCT:

Microcomputed tomography

ELISA:

Enzyme-linked immunoassay

H&E:

Hematoxylin and eosin

TRAP:

Tartrate-resistant acid phosphatase

GFP:

Green fluorescent protein

NBP:

Nitrogen-containing bisphosphonate

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Acknowledgments

Breast Cancer Campaign, UK, generously supported this study. We would also like to thank Mr Ken Mann for performing the RANKL immunohistochemistry and Dr Simon Cross for his help with the evaluation of the staining.

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Authors and Affiliations

  1. Academic Unit of Clinical Oncology, DU10, Medical School, University of Sheffield, Beech Hill Road, Sheffield, S10 2RX, UK

    H. K. Brown, P. D. Ottewell, C. A. Evans & I. Holen

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  1. H. K. Brown
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  2. P. D. Ottewell
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  3. C. A. Evans
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  4. I. Holen
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Correspondence to H. K. Brown.

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10585_2012_9481_MOESM1_ESM.ppt

Supplementary figure 1: (A) µCT trabecular bone measurements were carried out for the proximal tibia and distal femur in non-tumour bearing bones vs. bones from naïve mice. Data are expressed as % BV/TV (bone volume/tissue volume). (B) Sera from the entire study were collected from starved animals. PINP serum levels were measured by ELISA (Rat/Mouse PINP EIA, IDS) on n=6 day 10, n=9 day 15, n=8 day 19, n=10 day 24 and n=6 EOP, naïve n=5/time point. (C) CTX serum levels were measured by ELISA (RatLaps™ EIA for CTX, IDS) on a subset of animals from the study. Day 10 n=1, day 15 n=3, day 19 n=2, day 24 n=3, EOP n=2, naïve n=2/time point. (D) Mouse RANKL serum levels were measured by ELISA (RANKL Immunoassay, Biomedica) on tumour bearing animals with n=4 at day 10, n=7 at day 15, n=5 at day 19, n=6 at day 24 and n=3 at EOP, naïve n=3/time point. (E) Sclerostin serum levels were measured by ELISA (Sclerostin Immunoassay, R&D Systems) with n numbers as in (D). Mann–Whitney test for all time points separately, * is p<0.05 (PPT 636 kb)

10585_2012_9481_MOESM2_ESM.pptx

Supplementary table 1: Relative expression of bone-related genes measured by real-time reverse transcription-PCR and normalised to GAPDH. TaqMan® assays have been tested for species specificity. In vivo subcutaneous samples (n=8) and osseous samples (n=4) are compared to in vitro control cells (MDA-MB-231-GFP, n=3). * vs. in vitro control p<0.05, ^ vs. subcutaneous tumour p<0.05. Fold changes in bold indicate bone specific changes (PPTX 56 kb)

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Brown, H.K., Ottewell, P.D., Evans, C.A. et al. Location matters: osteoblast and osteoclast distribution is modified by the presence and proximity to breast cancer cells in vivo. Clin Exp Metastasis 29, 927–938 (2012). https://doi.org/10.1007/s10585-012-9481-5

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  • DOI: https://doi.org/10.1007/s10585-012-9481-5

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