Zoledronic acid treatment did not reduce establishment of metastases in RM1(BM) injected mice.

<p>(A) Total number of metastases, (B) bone-metastases, or (C) soft tissue metastases as assessed by one-way ANOVA. Each point represents the number of metastases in an individual mouse, bars indicate the median within the group.</p

Histological analysis with tetrachrome stain for effects of zoledronic acid-treatment on normal and tumour-bearing bones.

<p>Tetrachrome stain for differentiation of mineralised and unmineralised bone at 120x magnification Normal (A), or RM1(BM) tumour bearing bones (B–D). (A&B) Untreated, (C) 20 µg/kg ZOL (D) 100 µg/kg ZOL. T = tumour, Arrow = Trabecular bone.</p

Zoledronic acid preserves bone structure.

<p>MicroCT scans of mouse femur and tibia; normal control (A), RM1(BM) tumour-bearing (B), RM1(BM) tumour-bearing treated with the 20 µg/kg ZOL regimen (C), RM1(BM) tumour-bearing treated with the 100 µg/kg ZOL regimen (D).</p

Histological analysis of the effects of zoledronic acid-treatment on normal and tumour-bearing bones.

<p>H&E stained sections of the femoral head at 40X (A, C, E and G) and 120X (B, D, F and H) magnification showing normal (A&B) and RM1(BM) tumour bearing bones (C–H); Typical examples of bones from untreated mice (A–D) and mice treated with 20 µg/kg ZOL (E&F) or 100 µg/kg ZOL (G&H) are shown. T = tumour, Arrow = Trabecular bone.</p

Zoledronic acid treatment alters bone volume and the bone surface-area:volume ratio in normal and tumour-bearing bones.

<p>There was no significant change in bone surface area (A) induced by the presence of a tumour or treatment with zoledronic acid. However, bone volume was dramatically reduced in RM1(BM) containing bones and the treatment with ZOL prevented this loss in a dose-dependent manner (B), resulting in lower surface area/volume ratio (increased bone density) compared to normal or tumor-bearing bones (C). Results are based on measurements obtained from CT scans as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019389#s4" target="_blank">Materials and Methods</a> section. Statistical analysis was performed by one-way ANOVA followed by Tukey's post test, * p<0.05, ** p<0.01, *** p<0.001.</p

Zoledronic acid treatment improves mouse survival but does not reduce the number of metastases.

<p>(A) Kaplan-Meier survival plot of mice given an intra-arterial injection of RM1(BM) cells followed by treatment with the 100 µg/kg or 20 µg/kg zoledronic dosing regimens or vehicle only as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019389#s4" target="_blank">Materials and Methods</a>. The log rank test for trend indicates a trend of increased survival with increasing dose (p = 0.012), and a significant difference in survival between mice in the 100 µg/kg dose group vs vehicle control groups (p = 0.031, Kaplan-Meier followed by Breslow pair-wise comparison using SPSS 17.0).</p

Surrogate markers of bone metabolism in serum of normal, tumor-bearing and zoledronic acid treated mice.

<p>(A) Levels of the osteoblast marker, osteocalcin were reduced in serum of zoledronic acid treated mice. (B<i>)</i> Tartrate-resistant acid phosphatase 5b, an indicator of osteoclast activity, was also reduced with treatment. Points on the graph represent serum levels for individual mice while bars show the position of the means within each group. Statistical analysis was performed using one-way ANOVA for analysis followed by Tukey's post test. *** p<0.001.</p

LNCaP microaggregates are more resistant to docetaxel compared to cells grown as monolayers.

<p>LNCaP cells (50,000 cells/well) were treated with docetaxel over 48 hrs (A) or 72 hrs (B) at day 2 of growth either in 2D and 3D. Alamar blue was used to assess cell viability. Mean +/- SE, n = 4 biological replicates *P<0.05; the data shown is representative of three independent experiments. (C) Phase contrast images show the effects of docetaxel after 72 hrs on the morphology of the microaggregates (3D) compared to cells grown in monolayer (2D).</p

Apoptotic core can be controlled for using differently sized microwells.

<p>(A) Cleaved caspase-3 (CASP3; green), an apoptosis marker, was used to stain sections of RWPE-1, RWPE-2 and LNCaP microaggregates. (B) Due to the absence of cleaved caspase-3 in the small LNCaP aggregates, a large microwell (800 µm ×800 µm ×800 µm) was used to create large LNCaP microaggregates with an apoptotic core (CASP3 Lge). (C) The diameter of the LNCaP microaggregates (Sml) was compared with LNCaP microaggregates grown in the large microwells (Lge). A minimum of 50 aggregates were measured per condition. (D) Ki67 (green) was also used to stain proliferating cells within the large LNCaP aggregate. Nuclei were stained with DAPI (magenta); scale bar is 100 µm.</p

The PDMS microwell system produces prostate cancer cell aggregates that are viable and of a controlled size.

<p>(A) The diameters of LNCaP and RWPE-2 cell aggregates were measured over 14 days; mean +/- SD, n = 50 from *p<0.05, a paired students t-test was used to show significant differences in diameter at day 7 and 14. (B) FDA/PI stain was used to stain viable cells green and dead cells red on the days shown and the percentage area of dead cells per microaggregate has been quantified to show that the non-cancer prostate cells (RWPE-1) have greater cell death (percentage red pixels) in the microwell inserts compared to the prostate cancer cells (LNCaP and RWPE-2); mean +/- SD, n = 10. A paired students t-test was used to calculate significance*p<0.05. (C) The confocal images and phase contrast images (Day 14 Phase) show that prostate cancer cell lines (RWPE-2 and LNCaP) grow as compact smooth microaggregates until day 14. Conversely, the non-cancer cells (RWPE-1) form dispersed loose clusters which are non-viable and with no definable diameter at day 14. Scale bar is 100 µm.</p