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

Experimental design 12-week old female

Balb/c-nude mice with disseminated MDA-MB-231 breast tumour cells in bone underwent sham operation or ovariectomy (OVX), mimicking the pre- and post-menopausal bone microenvironment, respectively. To determine the effects of bone-targeted therapy, sham/OVX animals received saline or 100ug/kg ZOL weekly. Tumour growth was assessed by i n vivo imaging and effects on bone by RT-PCR, microCT, histomorphometry and measurements of bone markers. Disseminated tumour cells were detected by two-photon microscopy.

Materials and Methods Cell Culture

Low passage (< P10) human breast cancer cells, MDA-MB-231-luc2 tdTomato (authenticated and purchased from Caliper Lifer Sciences, Cheshire, UK) or MDA-MB-231 (European Collection of Cell Cultures, Wiltshire, UK) transfected with eGFP were used. Prior to in vivo inoculation eGFP expressing cells were incubated for 15 minutes with 25μM of 1,1′-Dioctadecyl-′, 3′-Tetramethylindodicarbocyanine, 4-Chlorobenzenesulfonate (DiD) (Life Technologies, Paisley, UK). Tumour growth was monitored using an IVIS (luminol) system (Caliper Life Sciences) (luc2) or an Illumatool Lighting System (LightTools Research, Encintas, CA) (eGFP). In vivo studies We used 12-week-old female Balb/c nude mice (Charles River, Kent, UK). Experiments were carried out in accordance with local guidelines and with Home Office approval under project licence 40/3462, University of Sheffield, UK. Following ovariectomy (OVX) or sham operation animals were sacrificed 1-8 weeks later (n=5/group) and bone effects assessed. Effects of OVX on tumour cell homing and colonisation of bone were established by OVX/sham operating mice 7 days before tumour cell inoculation (n=10/group). For studies of bone homing and colonisation mice were culled 24h and 8 weeks after tumour cell injection, respectively. Effects of OVX on disseminated tumour cells were assessed following injection of breast cancer cells 7 days before OVX, sham, or non-operation (n=10/group) in ER+ve and ER-ve cell lines. 1×10 5 DiD labelled MDA-MB-231-luc-2 tdTomato cells were injected into the left cardiac ventricle (i.c.), tumour growth was monitored for 8 weeks. 0.1mls saline or 1×10 5 MCF7 cells were injected i.c. 4 days following sham or implantation of 17β estradiol pellet (innovative Research of America). Effects of estradiol alone were assessed at 4 weeks and tumour growth monitored for 10 weeks. Effects of ZOL on OVX-induced tumour growth were investigated in mice inoculated with 1×10 5 DiD labelled MDA-MB-231-eGFP cells i.c., and given weekly ZOL (100 μg/kg) or saline (n=20/group) from day 5. Seven days following tumour cell inoculation, animals from both groups underwent either sham or OVX (n=10). Assessment of PTH on tumour growth was investigated in mice inoculated with 1×10 5 MDA-MB-231-luc-2 tdTomato cells i.c., and given PTH (80μg/kg) or saline daily for 5 days (n=10/group) and animals sacrificed at 5 weeks. Serum was stored at −80°C for ELISA, tibiae and femurs were fixed in 4% PFA for μCT analysis before decalcification in 1%PFA/0.5% EDTA and processing for histology. Bones for two-photon analysis were stored in OCT at −80°C.

Microcomputed tomograpgy imaging

Microcomputed tomography analysis was carried out using a Skyscan 1172 x-ray-computed microtomography scanner (Skyscan, Aartselaar, Belgium) equipped with an x-ray tube (voltage, 49kV; current, 200uA) and a 0.5-mm aluminium filter. Pixel size was set to 5.86 μm and scanning initiated from the top of the proximal tibia as previously described ( 10 ). Bone histology and measurement of tumour volume Osteoclasts were detected by toluidine blue and tartate-resistant acid phosphatase (TRACP) staining as previously described ( 11 ). Osteoblasts were identified as mononuclear, cuboidal cells residing in chains along the bone surface. The number of osteoclasts/osteoblasts per millimeter of cortical-endosteal bone surface and trabecular bone surfaces and the proportion of bone surface occupied by osteoclasts/osteoblasts was determined using a Leica RMRB upright microscope and OsteoMeasure software (Osteometrics inc.) as previously described ( 12 ).

Two-photon microscopy

Tibiae were imaged using a multiphoton confocal microscope (LSM510 NLO upright; Zeiss, Cambridge, UK). DiD labelled cells were visualised using a 900nm Chameleon laser, bone was detected using the 633nm multiphoton laser (Coherent, Santa Clara, CA.) and images were reconstructed in LSM software version 4.2 (Zeiss).

Biochemical analysis

Serum concentrations of TRACP 5b, P1NP and PTH were measured using commercially available ELISA kits: MouseTRAP™ Assay (Immunodiagnostic systems), Rat/Mouse P1NP competitive immunoassay kit (Immunodiagnostic Systems) and Mouse PTH (Uscn Life Sciences), respectively Real-time PCR Gene expression was analysed on 3 custom-made microarray plates (Ref: 0186817032) per group (all reagents from Applied Biosystems, Warrington UK): Relative mRNA expression compared with the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Hs99999905_m1) was assessed using an ABI 7900 PCR System (Perkin Elmer, Foster City, CA) and Taqman universal master mix). Fold change in gene expression between treatment groups was assessed by directly inserting CT values into Data Assist V3.01 software (Applied Biosystems) and changes in gene expression were only analysed for genes with a CT value of ≤25.

Statistical Analysis

Statistical analysis was by one way analysis of variance (ANOVA) followed by Newman-Keuls multiple comparison test. Statistical significance was defined as P less than or equal to 0.05. All P values are two-sided.

Supplementary Material Supplementary Tables 1-3 Supplementary Figure 1 Supplementary Figure 2 Supplementary Figure 3 Supplementary Figure 4