Anoja Giles

Ultrasound Imaging of Apoptosis in Tumor Response: Novel Preclinical Monitoring of Photodynamic Therapy Effects

High-frequency ultrasound is a novel method to detect apoptotic cell death based on changes in cell morphology that cause alterations in the viscoelastic and, consequently, the acoustic properties of cell ensembles and tissues. In this study, we evaluated the first preclinical tumor-based use of high-frequency ultrasound spectroscopy to noninvasively monitor tumor treatment by following xenograft malignant melanoma tumor responses to photodynamic therapy (PDT) in vivo. We observed a time-dependant increase in ultrasound backscatter variables after treatment. The observed increases in spectroscopic variables correlated with morphologic findings, indicating increases in apoptotic cell death, which peaked at 24 hours after PDT. We analyzed the changes in spectral slope and backscatter in relation to apoptosis and histologic variations in cell nuclear size. Changes in spectral slope strongly correlated with the changes in mean nuclear size over time, associated with apoptosis, after PDT (P < 0.05). At 48 hours, a decrease in ultrasound backscatter was observed, which could be explained by an increase in cell nuclear degradation. In summary, we show that high-frequency ultrasound spectroscopic variables can be used noninvasively to monitor response after treatment in a preclinical tumor cancer model. These findings provide a foundation for future investigations regarding the use of ultrasound to monitor and aid the customization of treatments noninvasively based on responses to specific interventions.

Tumor radiation response enhancement by acoustical stimulation of the vasculature

We have discovered that ultrasound-mediated microbubble vascular disruption can enhance tumor responses to radiation in vivo. We demonstrate this effect using a human PC3 prostate cancer xenograft model. Results indicate a synergistic effect in vivo with combined single treatments of ultrasound-stimulated microbubble vascular perturbation and radiation inducing an over 10-fold greater cell kill with combined treatments. We further demonstrate with experiments in vivo that induction of ceramide-related endothelial cell apoptosis, leading to vascular disruption, is a causative mechanism. In vivo experiments with ultrasound and bubbles permit radiation doses to be decreased significantly for comparable effect. We envisage this unique combined ultrasound-based vascular perturbation and radiation treatment method being used to enhance the effects of radiation in a tumor, leading to greater tumor eradication.

Quantitative Ultrasound Characterization of Responses to Radiotherapy in Cancer Mouse Models

Purpose: Currently, no imaging modality is used routinely to assess tumor responses to radiotherapy within hours to days after the delivery of treatment. In this study, we show the application of quantitative ultrasound methods to characterize tumor responses to cancer radiotherapy in vivo, as early as 24 hours after treatment administration. Experimental Design: Three mouse models of head and neck cancer were exposed to radiation doses of 0, 2, 4, and 8 Gray. Data were collected with an ultrasound scanner using frequencies of 10 to 30 MHz. Ultrasound estimates calculated from normalized power spectra and parametric images (spatial maps of local estimates of ultrasound parameters) were used as indicators of response. Results: Two of the mouse models (FaDu and C666-1) exhibited large hyperechoic regions at 24 hours after radiotherapy. The ultrasound integrated backscatter increased by 6.5 to 8.2 dB (P < 0.001) and the spectral slopes increased from 0.77 to 0.90 dB/MHz for the C666-1 tumors and from 0.54 to 0.78 dB/MHz for the FaDu tumors (P < 0.05), in these regions compared with preirradiated tumors. The hyperechoic regions in the ultrasound images corresponded in histology to areas of cell death. Parametric images could discern the tumor regions that responded to treatment. The other cancer mouse model (Hep-2) was resistant to radiotherapy. Conclusions: The results indicate that cell structural changes after radiotherapy have a significant influence on ultrasound spectral parameters. This provides a foundation for future investigations regarding the use of ultrasound in cancer patients to individualize treatments noninvasively based on their responses to specific interventions.

Quantitative Ultrasound Characterization of Cancer Radiotherapy Effects In Vitro

PURPOSE Currently, no routinely used imaging modality is available to assess tumor responses to cancer treatment within hours to days after radiotherapy. In this study, we demonstrate the preclinical application of quantitative ultrasound methods to characterize the cellular responses to cancer radiotherapy in vitro. METHODS AND MATERIALS Three different cell lines were exposed to radiation doses of 2-8 Gy. Data were collected with an ultrasound scanner using frequencies of 10-30 MHz. As indicators of response, ultrasound integrated backscatter and spectral slope were determined from the cell samples. These parameters were corrected for ultrasonic attenuation by measuring the attenuation coefficient. RESULTS A significant increase in the ultrasound integrated backscatter of 4-7 dB (p < 0.001) was found for radiation-treated cells compared with viable cells at all radiation doses. The spectral slopes decreased in the cell samples that predominantly underwent mitotic arrest/catastrophe after radiotherapy, consistent with an increase in cell size. In contrast, the spectral slopes did not change significantly in the cell samples that underwent a mix of cell death (apoptosis and mitotic arrest), with no significant change in average cell size. CONCLUSION The changes in ultrasound integrated backscatter and spectral slope were direct consequences of cell and nuclear morphologic changes associated with cell death. The results indicate that this combination of quantitative ultrasonic parameters has the potential to assess the cell responses to radiation, differentiate between different types of cell death, and provide a preclinical framework to monitor tumor responses in vivo.

High-frequency ultrasound for monitoring changes in liver tissue during preservation

Currently the only method to assess liver preservation injury is based on liver appearance and donor medical history. Previous work has shown that high-frequency ultrasound could detect ischemic cell death due to changes in cell morphology. In this study, we use high-frequency ultrasound integrated backscatter to assess liver damage in experimental models of liver ischemia. Ultimately, our goal is to predict organ suitability for transplantation using high-frequency imaging and spectral analysis techniques. To examine the effects of liver ischemia at different temperatures, livers from Wistar rats were surgically excised, immersed in phosphate buffer saline and stored at 4 and 20 degrees C for 24 h. To mimic organ preservation, livers were excised, flushed with University of Wisconsin (UW) solution and stored at 4 degrees C for 24 h. Preservation injury was simulated by either not flushing livers with UW solution or, before scanning, allowing livers to reach room temperature. Ultrasound images and corresponding radiofrequency data were collected over the ischemic period. No significant increase in integrated backscatter (approximately 2.5 dBr) was measured for the livers prepared using standard preservation conditions. For all other ischemia models, the integrated backscatter increased by 4-9 dBr demonstrating kinetics dependent on storage conditions. The results provide a possible framework for using high-frequency imaging to non-invasively assess liver preservation injury.

Low-frequency quantitative ultrasound imaging of cell death in vivo.

PURPOSE Currently, no clinical imaging modality is used routinely to assess tumor response to cancer therapies within hours to days of the delivery of treatment. Here, the authors demonstrate the efficacy of ultrasound at a clinically relevant frequency to quantitatively detect changes in tumors in response to cancer therapies using preclinical mouse models. METHODS Conventional low-frequency and corresponding high-frequency ultrasound (ranging from 4 to 28 MHz) were used along with quantitative spectroscopic and signal envelope statistical analyses on data obtained from xenograft tumors treated with chemotherapy, x-ray radiation, as well as a novel vascular targeting microbubble therapy. RESULTS Ultrasound-based spectroscopic biomarkers indicated significant changes in cell-death associated parameters in responsive tumors. Specifically changes in the midband fit, spectral slope, and 0-MHz intercept biomarkers were investigated for different types of treatment and demonstrated cell-death related changes. The midband fit and 0-MHz intercept biomarker derived from low-frequency data demonstrated increases ranging approximately from 0 to 6 dBr and 0 to 8 dBr, respectively, depending on treatments administrated. These data paralleled results observed for high-frequency ultrasound data. Statistical analysis of ultrasound signal envelope was performed as an alternative method to obtain histogram-based biomarkers and provided confirmatory results. Histological analysis of tumor specimens indicated up to 61% cell death present in the tumors depending on treatments administered, consistent with quantitative ultrasound findings indicating cell death. Ultrasound-based spectroscopic biomarkers demonstrated a good correlation with histological morphological findings indicative of cell death (r2=0.71, 0.82; p<0.001). CONCLUSIONS In summary, the results provide preclinical evidence, for the first time, that quantitative ultrasound used at a clinically relevant frequency, in addition to high-frequency ultrasound, can detect tissue changes associated with cell death in vivo in response to cancer treatments.

Bioeffects of Ultrasound-Stimulated Microbubbles on Endothelial Cells: Gene Expression Changes Associated with Radiation Enhancement In Vitro

Ultrasound can be used to target endothelial cells in cancer therapy where the destruction of vasculature leads to tumor cell death. Here, we demonstrate ultrasound bioeffects in which the levels of genes in endothelial cells can be significantly altered by ultrasound-stimulated microbubble exposure. These were compared with established effects of radiation on endothelial cells at a gene level. Human-endothelial cells were exposed to ultrasound and microbubbles, radiation or combinations of ultrasound, microbubbles and radiation. Gene expression analyses revealed an up-regulation of genes known to be involved in apoptosis and ceramide-induced apoptotic pathways, including SMPD2, UGT8, COX6B1, Caspase 9 and MAP2K1 with ultrasound-stimulated microbubble exposure but not SMPD1. This was supported by immunohistochemistry and morphologic changes examined with cell microscopy, which showed changes in SMPD1 gene product in cells with microbubble exposure. This supports the hypothesis that ultrasound-stimulated microbubbles can induce significant bioeffect-related changes in gene expression and can affect ceramide signaling pathways in endothelial cells, leading to apoptosis.

Detection of Apoptotic Cell Death In Vitro in the Presence of Gd-DTPA-BMA

Due to variability in patient response to cancer therapy, there is a growing interest in monitoring patient progress during treatment. Apoptotic cell death is one early marker of tumor response to treatment. Using known extracellular concentrations of gadolinium diethylenetriamine pentaacetic acid bismethylamide (Gd‐DTPA‐BMA) to vary the exchange regime, T1 and T2 relaxation data for acute myeloid leukemia (AML) cell samples were obtained and then analyzed using a two‐pool model of relaxation with exchange. Leukemia cells treated with cisplatin to induce apoptosis exhibited a statistically significant (P < 0.05) decrease in intracellular longitudinal relaxation time, T1I, from 1030 ms to 940 ms, a decrease (P < 0.001) in the intracellular water fraction, M0I, from 0.86 to 0.68 and a statistically significant increase (P < 0.01) in transmembrane water exchange rate, kIE, from 1.4 s−1 to 6.8 s−1. The changes in MR parameters correlated with quantitative histology, such as cellular cross‐sectional area and average nuclear area measurements. The results of this study emphasize the importance of accounting for water exchange in dynamic contrast‐enhanced MRI (DCE‐MRI) studies, particularly those that examine tumor response to therapies in which apoptotic changes occur. Magn Reson Med, 2009.

Microbubble and ultrasound radioenhancement of bladder cancer

Background:Tumour vasculature is an important component of tumour growth and survival. Recent evidence indicates tumour vasculature also has an important role in tumour radiation response. In this study, we investigated ultrasound and microbubbles to enhance the effects of radiation.Methods:Human bladder cancer HT-1376 xenografts in severe combined immuno-deficient mice were used. Treatments consisted of no, low and high concentrations of microbubbles and radiation doses of 0, 2 and 8 Gy in short-term and longitudinal studies. Acute response was assessed 24 h after treatment and longitudinal studies monitored tumour response weekly up to 28 days using power Doppler ultrasound imaging for a total of 9 conditions (n=90 animals).Results:Quantitative analysis of ultrasound data revealed reduced blood flow with ultrasound-microbubble treatments alone and further when combined with radiation. Tumours treated with microbubbles and radiation revealed enhanced cell death, vascular normalisation and areas of fibrosis. Longitudinal data demonstrated a reduced normalised vascular index and increased tumour cell death in both low and high microbubble concentrations with radiation.Conclusion:Our study demonstrated that ultrasound-mediated microbubble exposure can enhance radiation effects in tumours, and can lead to enhanced tumour cell death.

High frequency ultrasound imaging of changes in cell structure including apoptosis

It has been previously shown that high frequency ultrasound (20-100 MHz) can be used to detect cellular structure changes in tissues and cell ensembles. Using spectral analysis methods to analyze radio-frequency data collected from in vitro and in vivo models, the changes seen during apoptotic cell death are very striking. Imaging changes in cell structure has implications in a broad range of fields, from cancer treatment monitoring to organ transplantation. However, the changes seen in the backscattered ultrasound intensity and frequency spectrum are not fully understood. In this paper we propose and explore a model for studying how the changes in the sizes, spatial distribution, and acoustic impedance of the scattering sources within the cells are related to the resulting backscattered ultrasound signal.