Focused Ultrasound Stimulation of Microbubbles in Combination With Radiotherapy for Acute Damage of Breast Cancer Xenograft Model

Objective: Several studies have focused on the use of ultrasound-stimulated microbubbles (USMB) to induce vascular damage in order to enhance tumor response to radiation. Methods: In this study, power Doppler imaging was used along with immunohisto- chemistry to investigate the effects of combining radiation therapy (XRT) and USMB using an ultrasound-guided focused ultrasound (FUS) therapy system in a breast cancer xenograft model. Specifically, MDA-MB-231 breast cancer xenograft tumors were induced in severe combined immuno-deficient female mice. The mice were treated with FUS alone, ultrasound and microbubbles (FUS + MB) alone, 8 Gy XRT alone, or a combined treatment consisting of ultrasound, microbubbles, and XRT (FUS + MB + XRT). Power Doppler imaging was conducted before and 24 h after treatment, at which time mice were sacrificed and tumors assessed histolog- ically. The immunohistochemical analysis included terminal deoxynucleotidyl transferase dUTP nick end labeling, hematoxylin and eosin, cluster of differentiation-31 (CD31), Ki-67, carbonic anhydrase (CA-9), and ceramide labeling. Results: Tumors receiving treat- ment of FUS + MB combined with XRT demonstrated significant increase in cell death (p = 0.0006) compared to control group. Furthermore, CD31 and Power Doppler analysis revealed reduced tumor vascularization with combined treatment indicating (P \u3c .0001) and (P = .0001), respectively compared to the control group. Additionally, lesser number of proliferating cells with enhanced tumor hypoxia, and ceramide content were also reported in group receiving a treatment of FUS + MB + XRT. Conclusion: The study results demonstrate that the combination of USMB with XRT enhances treatment outcomes

Dll4-notch signalling blockade synergizes combined ultrasound-stimulated microbubble and radiation therapy in human colon cancer xenografts.

Tumour vasculature acts as an essential lifeline for tumour progression and facilitates metastatic spread. Novel vascular targeting strategies aiming to sustain vascular shutdown could potentially induce substantial damage, resulting in a significant tumour growth delay. We investigated the combination of two novel complementary vascular targeting agents with radiation therapy in a strategy aiming to sustain vascular disruption. Experiments were carried out with delta-like ligand 4 (Dll4) blockade (angiogenesis deregulator) treatment administered in combination with a radiation-based vascular destruction treatment in a highly aggressive well-perfused colon cancer tumour line implanted in female athymic nude mice. Tumours were treated with permutations of radiation, ultrasound-stimulated microbubbles (USMB) and Dll4 monoclonal antibody (mAb). Tumour vascular response was assessed with three-dimensional power Doppler ultrasound to measure active flow and immunohistochemistry. Tumour response was assessed with histochemical assays and longitudinal measurements of tumour volume. Our results suggest a significant tumour response in animals treated with USMB combined with radiation, and Dll4 mAb, leading to a synergistic tumour growth delay of up to 24 days. This is likely linked to rapid cell death within the tumour and a sustained tumour vascular shutdown. We conclude that the triple combination treatments cause a vascular shutdown followed by a sustained inhibition of angiogenesis and tumour cell death, leading to a rapid tumour vascular-based 'collapse' and a significant tumour growth delay

Optimization of microbubble enhancement of hyperthermia for cancer therapy in an in vivo breast tumour model.

We have demonstrated that exposing human breast tumour xenografts to ultrasound-stimulated microbubbles enhances tumour cell death and vascular disruption resulting from hyperthermia treatment. The aim of this study was to investigate the effect of varying the hyperthermia and ultrasound-stimulated microbubbles treatment parameters in order to optimize treatment bioeffects. Human breast cancer (MDA-MB-231) tumour xenografts in severe combined immunodeficiency (SCID) mice were exposed to varying microbubble concentrations (0%, 0.1%, 1% or 3% v/v) and ultrasound sonication durations (0, 1, 3 or 5 min) at 570 kPa peak negative pressure and central frequency of 500 kHz. Five hours later, tumours were immersed in a 43°C water bath for varying hyperthermia treatment durations (0, 10, 20, 30, 40, 50 or 60 minutes). Results indicated a significant increase in tumour cell death reaching 64 ± 5% with combined treatment compared to 11 ± 3% and 26 ± 5% for untreated and USMB-only treated tumours, respectively. A similar but opposite trend was observed in the vascular density of the tumours receiving the combined treatment. Optimal treatment parameters were found to consist of 40 minutes of heat with low power ultrasound treatment microbubble parameters of 1 minute of sonification and a 1% microbubble concentration

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