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

Development of Quantitative Optical Coherence Tomography Methods for Cell Death Detection

Current clinical cancer treatment monitoring methods rely largely on measurements of tumour volume to assess treatment efficacy several weeks to months after the start of therapy. The development of early monitoring methods to determine a patientâ s response within the first days of a new treatment could significantly impact the overall outcome. Treatment induced cancer cell death occurs before changes in tumour volume. Cell death is characterized by a series of predictable and organized subcellular morphological changes. Light scattering theory predicts that changes in the size, shape, number density and spatial organization of subcellular structures will affect light backscattered from tissues. The objective of this thesis was to develop quantitative methods using optical coherence tomography (OCT) to non-invasively detect cell death in tissues undergoing cancer therapy. Data acquisition and analysis methods were designed to measure variations in the OCT signal resulting from morphological changes in dying cells. Specifically, parameters were developed to measure changes in the speckle intensity statistics, spectral characteristics and the temporal intensity fluctuations of the OCT signal. In a series of in vitro experiments it was demonstrated that the quantitative OCT parameters are sensitive to morphological changes related to cell death. Measured changes in the quantitative parameters 24 to 48 hours after the induction of cell death were linked to structural changes observed in the cell nucleus using light microscopy. Based on the known kinetics of cell death, early variations in the spectral parameters of the OCT signal and decorrelation rate of the speckle intensities were linked to mitochondrial morphology. Finally, preliminary studies using mouse tumour models demonstrated the feasibility of using these parameters to detect cell death in vivo.Ph.D

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

Ultrasound microbubble potentiated enhancement of hyperthermia-effect in tumours.

It is now well established that for tumour growth and survival, tumour vasculature is an important element. Studies have demonstrated that ultrasound-stimulated microbubble (USMB) treatment causes extensive endothelial cell death leading to tumour vascular disruption. The subsequent rapid vascular collapse translates to overall increases in tumour response to various therapies. In this study, we explored USMB involvement in the enhancement of hyperthermia (HT) treatment effects. Human prostate tumour (PC3) xenografts were grown in mice and were treated with USMB, HT, or with a combination of the two treatments. Treatment parameters consisted of ultrasound pressures of 0 to 740 kPa, the use of perfluorocarbon-filled microbubbles administered intravenously, and an HT temperature of 43°C delivered for various times (0-50 minutes). Single and multiple repeated treatments were evaluated. Tumour response was monitored 24 hours after treatments and tumour growth was monitored for up to over 30 days for a single treatment and 4 weeks for multiple treatments. Tumours exposed to USMB combined with HT exhibited enhanced cell death (p<0.05) and decreased vasculature (p<0.05) compared to untreated tumours or those treated with either USMB alone or HT alone within 24 hours. Deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and cluster of differentiation 31 (CD31) staining were used to assess cell death and vascular content, respectively. Further, tumours receiving a single combined USMB and HT treatment exhibited decreased tumour volumes (p<0.05) compared to those receiving either treatment alone when monitored over the duration of 30 days. Additionally, tumour response monitored weekly up to 4 weeks demonstrated a reduced vascular index and tumour volume, increased fibrosis and lesser number of proliferating cells with combined treatment of USMB and HT. Thus in this study, we characterize a novel therapeutic approach that combines USMB with HT to enhance treatment responses in a prostate cancer xenograft model in vivo

Microbubble-based enhancement of radiation effect: Role of cell membrane ceramide metabolism

<div><p>Ultrasound (US) stimulated microbubbles (MB) is a new treatment approach that sensitizes cancer cells to radiation (XRT). The molecular pathways in this response remain unelucidated, however, previous data has supported a role for cell membrane-metabolism related pathways including an up regulation of UDP glycosyltransferase 8 (UGT8), which catalyzes the transfer of galactose to ceramide, a lipid that is associated with the induction of apoptotic signalling. In this study, the role of UGT8 in responses of prostate tumours to ultrasound-stimulated microbubble radiation enhancement therapy is investigated. Experiments were carried out with cells <i>in vitro</i> and tumours <i>in</i> vivo in which UGT8 levels had been up regulated or down regulated. Genetically modified PC3 cells were treated with XRT, US+MB, or a combination of XRT+US+MB. An increase in the immunolabelling of ceramide was observed in cells where UGT8 was down-regulated as opposed to cells where UGT8 was either not regulated or was up-regulated. Clonogenic assays have revealed a decreased level of cellular survival with the down-regulation of UGT8. Xenograft tumours generated from stably transfected PC3 cells were also treated with US+MB, XRT or US+MB+XRT. Histology demonstrated more cellular damage in tumours with down-regulated UGT8 in comparison with control tumours. In contrast, tumours with up-regulated UGT8 had less damage than control tumours. Power Doppler imaging indicated a reduction in the vascular index with UGT8 down-regulation and photoacoustic imaging revealed a reduction in oxygen saturation. This was contrary to when UGT8 was up regulated. The down regulation of UGT8 led to the accumulation of ceramide resulting in more cell death signalling and therefore, a greater enhancement of radiation effect when vascular disruption takes place through the use of ultrasound-stimulated microbubbles.</p></div

Photoacoustic post-treatment images of xenograft PC3 modified tumours in vivo and oxygen saturation analyses.

<p>Percent change in oxygen saturation levels twenty-four hours after treatments is significantly different with the combined treatments, but not with the single treatments of sham, down regulated and up regulated tumours, where a reduction in the saturation is observed, with more reduction in the down regulated xenograft. Scale bar represents 1 mm.</p

Summary of UGT8 signalling and its effect on ceramide metabolism.

<p>When UGT8 levels are reduced, ceramide, either produced at the cellular membrane or in the endoplasmic reticulum, accumulates or initiates an apoptotic signal. However, when UGT8 levels are elevated, it adds a sugar moiety to ceramide in the Golgi apparatus, converting it to galactosylceramide, and this leads to the reduction of ceramide levels and possibly its apoptotic signalling, thus making cells more resistant to therapy.</p

TUNEL staining of xenograft PC3 modified tumours sections treated with various conditions.

<p>Control and sham tumours illustrate an increase in levels of apoptotic cell death which was significant in the combined treatments. A further increase in cell death is observed in tumours induced by cells stably transfected with anti-<i>UGT8</i> sh-RNA. In contrast, less cell death is observed with up-regulated <i>UGT8</i>. Scale bar represents 50 μm.</p

H & E stained sections from xenograft PC3 modified tumours not treated with combinations of MB/XRT.

<p>The upper two panels show low magnification whole tumour sections and the lower two panels illustrate high magnification images. Controls, not transfected, or transfected with scrambled shRNA (sham) demonstrated apoptotic cell death more with combined treatments and less with single treatments. However, tumours induced by cells stably transfected with anti-<i>UGT8</i> sh-RNA, show different types of cell death and fibrosis. Cell death was less obvious in xenograft tumours initiated from cells with up-regulated <i>UGT8</i>. Scale bar for low magnification images is 1mm and for high magnification represents 50 μm.</p

Photoacoustic post-treatment images of xenograft PC3 modified tumours in vivo and oxygen saturation analyses.

<p>Percent change in oxygen saturation levels twenty-four hours after treatments is significantly different with the combined treatments, but not with the single treatments of sham, down regulated and up regulated tumours, where a reduction in the saturation is observed, with more reduction in the down regulated xenograft. Scale bar represents 1 mm.</p