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

In vivo assessment of prostate cancer response using quantitative ultrasound characterization of ultrasonic scattering properties

Abstract Background The study here investigated quantitative ultrasound (QUS) parameters to assess tumour response to ultrasound-stimulated microbubbles (USMB) and hyperthermia (HT) treatment in vivo. Mice bearing prostate cancer xenografts were exposed to various treatment conditions including 1% (v/v) Definity microbubbles stimulated at ultrasound pressures 246 kPa and 570 kPa and HT duration of 0, 10, 40, and 50 min. Ultrasound radiofrequency (RF) data were collected using an ultrasound transducer with a central frequency of 25 MHz. QUS parameters based on form factor models were used as potential biomarkers of cell death in prostate cancer xenografts. Results The average acoustic concentration (AAC) parameter from spherical gaussian and the fluid-filled spherical models were the most efficient imaging biomarker of cell death. Statistical significant increases of AAC were found in the combined treatment groups: 246 kPa + 40 min, 246 kPa + 50 min, and 570 kPa + 50 min, in comparison with control tumours (0 kPa + 0 min). Changes in AAC correlates strongly (r2 = 0.62) with cell death fraction quantified from the histopathological analysis. Conclusion Scattering property estimates from spherical gaussian and fluid-filled spherical models are useful imaging biomarkers for assessing tumour response to treatment. Our observation of changes in AAC from high ultrasound frequencies was consistent with previous findings where parameters related to the backscatter intensity (AAC) increased with cell death

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

Quantitative Ultrasound for Evaluation of Tumour Response to Ultrasound-Microbubbles and Hyperthermia

Objectives: Prior study has demonstrated the implementation of quantitative ultrasound (QUS) for determining the therapy response in breast tumour patients. Several QUS parameters quantified from the tumour region showed a significant correlation with the patient's clinical and pathological response. In this study, we aim to identify if there exists such a link between QUS parameters and changes in tumour morphology due to combined ultrasound-stimulated microbubbles (USMB) and hyperthermia (HT) using the breast xenograft model (MDA-MB-231). Method: Tumours grown in the hind leg of severe combined immuno-deficient mice were treated with permutations of USMB and HT. Ultrasound radiofrequency data were collected using a 25 MHz array transducer, from breast tumour-bearing mice prior and post-24-hour treatment. Result: Our result demonstrated an increase in the QUS parameters the mid-band fit and spectral 0-MHz intercept with an increase in HT duration combined with USMB which was found to be reflective of tissue structural changes and cell death detected using haematoxylin and eosin and terminal deoxynucleotidyl transferase dUTP nick end labelling stain. A significant decrease in QUS spectral parameters was observed at an HT duration of 60 minutes, which is possibly due to loss of nuclei by the majority of cells as confirmed using histology analysis. Morphological alterations within the tumour might have contributed to the decrease in backscatter parameters. Conclusion: The work here uses the QUS technique to assess the efficacy of cancer therapy and demonstrates that the changes in ultrasound backscatters mirrored changes in tissue morphology

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