Illustration of posited USMB, XRT and Dll4 mAb effects on tumour endothelial cells.

<p>We posit that pre-treating tumours with ultrasound-activated microbubbles may first radiosensitize the cells via a biophysical process. Upon radiation delivery, endothelial cells would first undergo apoptosis, leading to tumour cell death. Continued delivery of an angiogenesis deregulator would prevent tumour reperfusion, serving as a maintenance therapy.</p

Power Doppler results.

<p>A) Representative images of three-dimensional maximum intensity projection of power Doppler signal at 24 hours post treatment, and quantified power Doppler at B) 24 hours and C) 7 days, are shown. Animals receiving Dll4 mAb alone had a decrease in flow signal (VI) 24 hours after treatment delivery; the VI dropped further 7 days after therapy. USMB+XRT caused a rapid decrease in power Doppler signal. However, the active blood volume returned to baseline levels by 7 days. A rapid VI decrease of almost 60% was observed for XRT and Dll4 mAb as well as triple combination conditions. However, the VI drop persisted to 7 days only in animals receiving the triple condition treatment. Each experimental condition is represents an average of 5 animals. Error bars represent one standard error of the mean. Statistically significant changes are marked with * (p≤0.05). The scale bar represents 2 mm.</p

Tumour growth results.

<p>A) Normalized tumour growth curves. A delayed tumour growth in all treatment conditions was noted. Tumour growth curves followed similar trends for the XRT alone, the Dll4 mAb alone, and the USMB+XRT treatments. Combined USMB+XRT+Dll4 mAb was observed to induce the greatest response, with a decrease in tumour size at 5 days after treatment, followed by growth inhibition lasting for nearly 15 days. B) Quantified tumour growth delay to reach two times the starting volume. Treatments with XRT and continued Dll4 mAb experienced a significant synergistic tumour growth delay of 14 days. Meanwhile, the triple combination treatment (USMB+XRT+Dll4 mAb) caused a tumour growth delay of 24 days. Statistically significant differences in tumour growth delay are indicated with * for p≤0.05.</p

High magnification light microscope images of PC-3 xenografts immuno-stained with cyclophilin A.

<p>Tumors treated with ultrasound pulses at 750-staining. Each panel demonstrates a representative region of cell death occurred in the tumor. Microbubble concentrations are given as 8 µL/kg, 80 µL/kg, and 1000 µL/kg. The scale bar represents 25 µm.</p

Average changes in midband-fit parameter.

<p>Each bar represents the mean of midband-fit values of five mouse-borne tumors (n = 5). The error bar indicates the standard error within the sample size. Statistical testing using 2-way ANOVA indicates the effects caused by the changes in both microbubble concentration and dose of radiation to be very significant for every graph (<i>p</i><0.0001). Each graph shows the average changes in midband-fit for varied microbubble concentration and radiation doses at a fixed ultrasound pressure: 250 kPa, 570 kPa, and 750 kPa.</p

Results of statistical analysis performed on the “<i>Quantification of normalized fraction of condensed or fragmented nuclei</i>” using one-tailed paired t-test.

<p>Results of statistical analysis performed on the “<i>Quantification of normalized fraction of condensed or fragmented nuclei</i>” using one-tailed paired t-test.</p

Results of statistical analysis performed on the “Quantification of Ceramide” using 2-way ANOVA without replication.

<p>Results of statistical analysis performed on the “Quantification of Ceramide” using 2-way ANOVA without replication.</p

Results of statistical analysis performed on the “<i>Average Changes in Midband-fit, 0-MHz Intercept, and Slope parameter</i>” using 2-way ANOVA without replication.

<p>Results of statistical analysis performed on the “<i>Average Changes in Midband-fit, 0-MHz Intercept, and Slope parameter</i>” using 2-way ANOVA without replication.</p

CD31 staining results.

<p>A) Representative images of CD31 staining of tumour cross-sections. Images are of four of the treatment conditions obtained at 24 hours and 7 days after therapy. The scale bar represents 100 µm. B) Normalized CD31 staining per ROI at 24 hours and C) at 7 days after treatment start. We noted a non-significant, but near doubling of CD31 staining in animals treated with Dll4 mAb only. There was a significant decrease in CD31 staining in animals treated with USMB+XRT as well as those treated with the triple treatment conditions. By 7 days after treatment, there was a significant increase in CD31 stained vessels for all animals receiving Dll4 mAb treatment, whether alone or in combination with USMB and/or XRT. Animals treated with XRT alone, or in combination with USMB, had quantified CD31 stained counts similar to control animals. Statistically significance is indicated with * indicating a p≤0.05.</p

Cell death results.

<p>A) Representative 24 hour H&E stained tumour cross-sections for specified treatment conditions at low magnification. The bottom row is of high-magnification images of representative H&E stained tumour cross-sections for the specified treatments. The top row scale bar represents 2 mm and the bottom row scale bar represents 50 µm. Quantified ISEL staining at B) 24 hours and C) 7 days. Significant (p≤0.05) cell death was observed in tumours treated with USMB+XRT+Dll4 mAb, while a non-significant amount of cell death is observed for animals treated with XRT and Dll4 mAb. At 7 days after initial treatment, we noted significant (p≤0.05) amounts of cell death in both the combined XRT+Dll4 mAb and USMB+XRT+Dll4 mAb treatment conditions. Statistical significance is indicated with * for p≤0.05.</p