PC3 implantations treated with zoledronic acid (ZA, n = 4) show a bone-protective effect compared to controls (n = 8).

<p>(A) MRI tumor volume and ADC determined at day 21 post-treatment-initiation shows a retardation of tumor growth and significantly lower ADC in the zoledronic acid treated animals. (B) PRM_HU+ bar plot shows significantly higher volume of bone that increased in density after treatment compared to controls. (C) PRM_HU- bar plot shows minimal loss of bone in the ZA-treated group, compared to progressively increasing bone loss in the controls. (D) Representative images for a control (top) and ZA-treated (bottom) mouse showing (from top to bottom) an isosurface, CT slice, PRM overlay, and PRM scatterplot from pre-treatment to 21 days post-treatment.</p

LAPC-9 tumors show slower mixed PRMHU+/- response with docetaxel treatment compared to PC3.

<p>(A) Time plots of tumor volume (solid line) and ADC (dashed line) show successful response to treatment (n = 3) as volume shrinkage and ADC increase. (B) PRM_HU+ bar plot over time shows more bone density increase in the docetaxel-treated group compared to controls (n = 3), significant on days 14 and 21. (C) PRM_HU- bar plot over time shows very little bone loss in the treated group compared to elevated bone mineral loss in the controls (though not significant in this study). (D) Representative images for a control (top) and docetaxel-treated (bottom) mouse showing (from top to bottom) an isosurface, CT slice, PRM overlay, and PRM scatterplot from pre-treatment to 21 days post-treatment.</p

PRMHU plots over time post-implantation compare un-treated bone changes in PC3 (diamonds, solid line, n = 4) to LAPC-9 (squares, dashed line, n = 6) implants as quantified by (A) PRMHU+ and (B) PRMHU-.

<p>PRMHU plots over time post-implantation compare un-treated bone changes in PC3 (diamonds, solid line, n = 4) to LAPC-9 (squares, dashed line, n = 6) implants as quantified by (A) PRMHU+ and (B) PRMHU-.</p

Results from clinical trial.

<p>(A) Representative PRM overlays show stable disease (top) and progressive disease (bottom) for PRMHU (left) and PRMADC+ (right). Blue represents regions of decreased value, red increased value, and green statistically unchanged value. (B) The bar plot shows significant differences (marked with *) between stable disease (SD, gray, n = 8) and progressive disease (PD, black, n = 4) groups in volume fractions of bone PRM results (labeled PRM_HU-, volume fraction of decreased attenuation at about 10 weeks post-treatment) and DW-MRI (volume fraction of increased ADC at about 2 weeks post-treatment).</p

A CONsolidated Standards of Reporting Trials (CONSORT) flow diagram of the overall study patient population recruitment and disposition over the course of study.

<p>A CONsolidated Standards of Reporting Trials (CONSORT) flow diagram of the overall study patient population recruitment and disposition over the course of study.</p

Intratibial PC3 tumor response to docetaxel (n = 6), radiation (IR, n = 6), or combination treatment (n = 6) shows an additive effect by anatomical and diffusion MRI compared to controls (n = 8).

<p>(A) Tumor volumes plotted over time (p-values in legend show significance versus controls at day 7) show greater cell kill in the combination group (circles, solid line) than either docetaxel (triangles, long-dashed line) or IR (diamonds, short-dashed line) treatment alone, and all treatments resulted in significant cell kill over controls (squares, dotted line). (B) Comparison of mean ADC change to PRM_ADC+ at day 7 post-treatment-initialization resulted in no significant difference between measurements, but slightly elevated PRM_ADC+ over mean ADC in the combination treatment (significant difference from controls: * (p<0.05)). (C) ADC color over overlays are shown in the left two columns for pre-treatment and day 7, and corresponding PRM_ADC overlay and scatterplot are shown on the right.</p

High throughput screening for inducers Caspase 3/7.

<p>Glosensor expressing 1833 or D54 cells (A and B respectively) were used to screen a library of 1,280 pharmacologically active compounds (LOPAC). Fold induction of bioluminescence signal intensity over values obtained from vehicle treated cells was plotted at maximal induction (mean ± SEM).</p

Assessing drug sensitivity of rare and transient cell populations.

<p>A) FACS analysis of dissociated D54 cells sorted into CD133⁺ and CD133⁻ populations, P3 represents the CD133 expressing cell population. B) to E) Bioluminescence assay of CD133⁺ and CD133⁻ sorted D54 cells incubated with 200 ng/ml TRAIL (B), 50 µM MNS (C), 50 µM MK886 (D) or 12.5 µM GW7647 (E). Bioluminescence was plotted as fold induction over values obtained from vehicle treated cells. Experiments were performed in triplicates and plotted as mean ± SEM. Paired t-test was performed for all experiments and * denotes p<0.05 value at indicated time points.</p

Validation of HTS hits.

<p>A) Bioluminescence activity of cells treated with MNS was measured at 12 hours post treatment and plotted as fold induction. Experiments were performed at least in triplicates (mean ± SEM). B) Representative western blots for Luciferase, cleaved Caspase 3 and PARP or β-Actin as loading control of D54 cells treated with (25 µM) MNS for 12 hrs. C) Bioluminescence activity of cells treated with increasing concentrations of CV3988 at 12 hours post treatment. Data are plotted as fold induction over values obtained from vehicle treated cells. Experiments were performed in triplicates (mean ± SEM). D and E). Bioluminescence activity was measured at various time points using 1833 (D) or D54 (E) cells treated with CV3988 (12.5 µM), Z-VAD (20 µM) or a combination of Z-VAD plus CV3988 for 24 hrs. Data are plotted as fold induction and experiments were performed in triplicates and plotted as mean ± SEM. F) Representative western blots of Luciferase, Caspase 3, PARP or β-actin were performed on lysates obtained from D54 cells. Cells were either treated with CV3988 (12.5 µM), pre-treated with Z-VAD (20 µM) or treated with Z-VAD and CV3988 for 12 hrs.</p

Utility of Caspase 3/7 GloSensor for assessment of cell death in cells.

<p>A) Schematic of the Caspase 3/7 GloSensor reporter containing an N-terminus coding for the C-Luc domain (358–544) of luciferase and a C-terminus coding for the N-Luc domain (4–354) of luciferase and a adjoining sequence, DEVD, the Caspase 3/7 recognition sequence. B) The functional basis of the reporter, wherein Caspase 3/7 mediated cleavage at the DEVD sequence results in release of the luciferase peptides and reconstitution of the enzymatic activity and an increase in luminescence signal. C) Bioluminescence analysis of cells treated with 200 ng/ml TRAIL. Data is plotted as fold induction standardized to values obtained from vehicle treated cells. D) Western blot for Caspase 3 cleavage using D54 cells treated with TRAIL for 6 hrs. β-Actin was used to standardize loading. E) Bioluminescence analysis of D54 cells treated with varying concentrations (25–100 ng/ml) of an agonist anti-Fas antibody. Data is plotted as fold induction over values obtained from vehicle treated cells at every hour. F) Bioluminescence analysis of cells treated with a pan-Caspase inhibitor Z-VAD (20 µM), 50 µM Docetaxel or with Z-VAD and Docetaxel combined. Data is plotted as fold induction. Experiments were performed at least in triplicates and mean values were plotted ± SEM.</p