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

Objectives To investigate whether timing and sequencing of ultrasound‐stimulated microbubbles (USMBs) and external beam radiotherapy (XRT) affect the treatment response in a preclinical prostate cancer model. Methods Prostate cancer xenografts were treated with ultrasound‐stimulated lipid microspheres before and after 8‐Gy XRT. Treatments were separated by 0, 3, 6, 12, and 24 hours, with 5 tumors per group. Tumor effects were evaluated by microvessel density (measured by CD31 staining), cell death (terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end‐labeling and hematoxylin–eosin staining), and hypoxia (carbonic anhydrase 9 staining). Results Administering USMBs 6 hours before XRT showed the maximum treatment effect using all 3 assays. At this time, the mean cell death index ± SD was 36% ± 10%, compared with 19% ± 4% for no separation between USMB treatment and XRT; the microvessel density was 9 ± 3 counts per field (19 ± 5 without separation); and the percentage of hypoxic cells was 10% ± 5% (21% ± 4%). The observed treatment effect was greater with USMBs before XRT than when administering XRT first, but these differences were not statistically significant. Conclusions The maximum tumor effect was observed with USMBs delivered 6 hours before XRT. The sequencing of treatment did not have a significant effect on the tumor response.

Gold nanoparticles (GNP) have been shown to highly absorb ionizing radiation compared to tissue. GNPs have also been shown to be high absorbers of non-ionizing radiation with a peak absorbance at a wavelength dependent on their shape and size. This study investigated radiation dose enhancement in PC3 cells when in the presence of gold nanorods (NR) and near infrared light (IR). The PC3 cells were incubated with either PEGylated NRs (PNR) or anti prostate stem cell antigen antibody with nuclear localization sequence peptide conjugated NRs (AbNR). They were exposed to near infrared light at a wavelength of 810 nm to achieve a temperature of 42 ºC to 43 ºC for 60 minutes. They were also exposed to 160 kVp x-rays. It was found that both targeted and non-targeted GNPs when exposed to radiation and near infrared light synergistically enhanced radiation dose. It was also found that AbNRs provide greater dose enhancement than PNRs.

PURPOSE:At present, a one-size-fits-all approach is typically used for cancer therapy in patients. This is mainly because there is no current imaging-based clinical standard for the early assessment and monitoring of cancer treatment response. Here, the authors have developed, for the first time, a complete computer-aided-prognosis (CAP) system based on multiparametric quantitative ultrasound (QUS) spectroscopy methods in association with texture descriptors and advanced machine learning techniques. This system was used to noninvasively categorize and predict cell death levels in fibrosarcoma mouse tumors treated using ultrasound-stimulated microbubbles as novel endothelial-cell radiosensitizers.METHODS:Sarcoma xenograft tumor-bearing mice were treated using ultrasound-stimulated microbubbles, alone or in combination with x-ray radiation therapy, as a new antivascular treatment. Therapy effects were assessed at 2-3, 24, and 72 h after treatment using a high-frequency ultrasound. Two-dimensional spectral parametric maps were generated using the power spectra of the raw radiofrequency echo signal. Subsequently, the distances between "pretreatment" and "post-treatment" scans were computed as an indication of treatment efficacy, using a kernel-based metric on textural features extracted from 2D parametric maps. A supervised learning paradigm was used to either categorize cell death levels as low, medium, or high using a classifier, or to "continuously" predict the levels of cell death using a regressor.RESULTS:The developed CAP system performed at a high level for the classification of cell death levels. The area under curve of the receiver operating characteristic was 0.87 for the classification of cell death levels to both low/medium and medium/high levels. Moreover, the prediction of cell death levels using the proposed CAP system achieved a good correlation (r = 0.68,  p < 0.001) with histological cell death levels as the ground truth. A statistical test of significance between individual treatment groups with the corresponding control group demonstrated that the predicted levels indicated the same significant changes in cell death as those indicated by the ground-truth levels.CONCLUSIONS:The technology developed in this study addresses a gap in the current standard of care by introducing a quality control step that generates potentially actionable metrics needed to enhance treatment decision-making. The study establishes a noninvasive framework for quantifying levels of cancer treatment response developed preclinically in tumors using QUS imaging in conjunction with machine learning techniques. The framework can potentially facilitate the detection of refractory responses in patients to a certain cancer treatment early on in the course of therapy to enable switching to more efficacious treatments.

Blood vessels within tumours represent a key component for cancer cell survival. Disruption of these vessels can be achieved by inducing vascular endothelial-cell apoptosis. Moreover, endothelial cell apoptosis has been proven to be enhanced by ceramide-increasing drugs. Herein, we introduce a novel therapeutic approach which uses ultrasound-stimulated microbubbles used in combination with radiation to cause a rapid accumulation of ceramide in endothelial cells in-vitro. We also test this modality directly with other cell types as a general method of killing cancer cells. Human umbilical vein endothelial cells (HUVEC), acute myeloid leukemia cells (AML), murine fibrosarcoma cells (KHT-C), prostate cancer cells (PC3), breast cancer cells (MDA-MB-231) and astrocytes were used to evaluate this mechanism of inducing cell death. Survival was measured by clonogenic assays, and ceramide content was detected using immunohistochemistry. Exposure of cell types to ultrasound-stimulated bubbles alone resulted in increases in ceramide for all cell types and survivals of 12 ± 2%, 65 ± 5%, 83 ± 2%, 58 ± 4%, 58 ± 3%, 18 ± 7% for HUVEC, AML, PC3, MDA, KHT-C and astrocyte cells, respectively. Results from selected cell types involving radiation treatments indicated additive treatment enhancements and increases in intracellular ceramide content one hour after exposure to ultrasound-activated microbubbles and radiation. Endothelial cell survival decreased from 8 ± 1% after 2 Gy of radiation treatment alone and from 12 ± 2% after ultrasound and microbubbles alone, to 1 ± 1% with combined treatment. In Asmase +/+ astrocytes, survival decreased from 56 ± 2% after 2 Gy radiation alone and from 17 ± 7% after ultrasound and microbubbles alone, to 5 ± 2% when combined. Using ASMase deficient astrocytes ( Asmase -/-) and Sphingosine-1-phosphate (S1P), we also demonstrate that ultrasound-activated microbubbles stimulate ASMase activity and ceramide production. These findings suggest that ultrasound-stimulated microbubbles could be used as a new biomechanical method to enhance the effects of radiation.

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.

Effectiveness of chemotherapy depends on the extent to which drug molecules penetrate tissues and cells to reach their intended target and limited by toxic side effects exerted by the therapeutic agent. The application of ultrasound and microbubbles has been shown to increase cell permeability and enhance intracellular delivery of cell-impermeable molecules, a process known as sonoporation. This work investigated the potential of ultrasound and microbubbles to enhance the therapeutic effect of a chemotherapeutic agent using an in vitro cell suspension system. The objectives were to measure clonogenic cell viability following treatment with ultrasound and microbubbles in the presence and absence of a chemotherapeutic agent, and investigate the effect of cell line, treatment order and acoustic pressure. Cells in suspension - breast cancer (MDA-MB-231) and prostate cancer (PC3) - were treated with ultrasound and microbubbles (USMB) at settings of 500 kHz pulse centre frequency, acoustic pressure (240 and 580 kPa peak negative pressure), 32 μs pulse duration, 3 kHz pulse repetition frequency, 30 s insonation time and 0.5% v/v of microbubbles (DA04, Artenga Inc.) in the presence and absence of chemotherapeutic agent docetaxel (Taxotere®). Cells were treated with docetaxel for two hours. The order of USMB and chemotherapy (CM) were varied. Following treatment, cell viability was assessed using clonogenic assay. Ultrasound and microbubbles combined with docetaxel increased MDA-MB-231 cell death by ~10 folds compared to chemotherapy alone. Clonogenic viability of ~2% was achieved with the combined treatment (CM+USMB at 240 kPa; chemotherapeutic agent was added prior to USMB treatment) compared to -20% with chemotherapy alone (0.01 nmol/mL). Whereas with USMB+CM (chemotherapeutic agent was added five minutes following termination of ultrasound), cell viability of ~5% was achieved. Comparable results were achieved at higher acoustic pressures. PC3 cell viability of ~55% was achieved at 0.5 nmol/mL compared to ~65% at 0.0001 nmol/mL docetaxel, indicating that MDA-MB-231 was more sensitive to docetaxel compared to PC3 cells. In PC3 cells, a viability of -40% was achieved with the combined treatment, independent of the treatment order, compared to chemotherapy (~55%) and USMB (-90% at Pneg=240 kPa) treatments. Ultrasound and microbubbles enhanced the therapeutic effect of docetaxel, a chemotherapeutic agent, with a more pronounced enhancement in the chemosensitive cell line (MDA-MB-231) compared to the chemoresistant cell line (PC3). Future work will investigate this effect in vivo.

Microbubbles are used as therapeutic agents to improve drug delivery across blood vessels and cell membranes. Recently, it was shown that the application of ultrasound and microbubbles can enhance the therapeutic effect of radiotherapy using in vitro and in vivo tumour models. This phenomenon depended on ultrasound, microbubble and x-ray exposure parameters. In this study, the effect of ultrasound-microbubble (US+MB) and ionizing radiation (XRT) treatment scheduling on cell viability and production of ceramide, an apoptosis messenger, was investigated in vitro. Human prostate cancer (PC3) and KHT-C murine fibrosarcoma cells in suspension were exposed to US+MB (f=500kHz; Pneg=580kPa; PD=32μs; PRF=3kHz; insonation time 30s; and 1% v/v microbubbles (DA04, Artenga Inc.)) and XRT (3 Gy single fraction). Timing between the treatments immediately (i.e. within 15 minutes), 3 and 6 hours and their order were varied. Following treatment, cell viability was assessed using clonogenic assay. Ceramide level within cells was quantified for up to 7.5 hours following treatment using immunohistochemistry and spectrophotometry. Ultrasound and microbubbles improved the therapeutic effect of radiotherapy in KHT-C and PC3 cells; cell death increased by ~3-5 folds with the combined treatment compared to each treatment alone. Maximal KHT-C cell death (70±2%) was achieved when US+MB was followed in three hours by XRT. KHT-C cell death of 43±2% and 25±2% was achieved with US+MB and XRT treatments, respectively. The maximal PC3 cell death (83±2%) was achieved when US+MB was followed by XRT immediately (i.e. within 15 minutes). PC3 cell death of 15±4% and 48±5% was achieved with US+MB and XRT treatments, respectively. US+MB and XRT treatments increased the level of ceramide in both cell lines compared to untreated controls. A correlation was found between cell death and ceramide at 3 h following treatment for the KHT-C and PC3 cells with - - correlation coefficients of 0.938 and 0.706, respectively. Ultrasonically-stimulated microbubbles can enhance the therapeutic effect of radiotherapy and the timing between the treatments and their order are important in the optimization of the therapeutic effect of radiotherapy. Future work will investigate this in vivo.

The aim of this work was to develop a novel ultrasound-microbubble mediated vascular-disruption based method to enhance the effects of ionizing radiation on tumours. In this study, we hypothesized that ultrasound-activated microbubbles can be used as radioenhancers in an in vitro cell line. Acute myeloid leukemia (AML-5) cells in suspension were exposed to ultrasound pulses and ionizing radiation. Variations in insonating acoustic pressure (125-570 kPa), Definity microbubble concentration (0-3% v/v), ionizing radiation dose (0-8 Gy) and treatment order of ultrasound and radiation were investigated. The effect on the viability of cells was assessed using flow cytometry with propidium iodide immediately and 48 hours following treatment. Clonogenic viability of cells was assessed using a clonogenic assay and electron microscopy images were acquired of untreated, treated with ultrasound and microbubbles, treated with ionizing radiation and with the combined treatments. The results indicate that ultrasound-activated microbubbles can enhance the effect of ionizing radiation in AML-5 cells. Cell death increased by ~35% with the combined treatment of ultrasound and ionizing radiation (42%?6%) compared to ultrasound (77%?5%) and radiotherapy (71%?7%) 48 hours following the treatments. The ability of ultrasound and microbubbles to enhance therapeutic effect of radiotherapy depended on ultrasound pressure and microbubble concentration. Higher acoustic pressures (570 kPa) and microbubble concentrations (greater than 1.5% v/v) were more effective at increasing cell death in the combined treatment. Cell death increased with ionizing radiation dose in treatments with radiotherapy only and with combined treatments. The induced enhancement was not sensitive to the order of the treatments with one hour between treatments. Clonogenic survival assay showed a reduced viability in the combined treatment (~2%) compared to ultrasound and microbubble treatment (~30%) and ionizing radiation (~11%). Microscopy images of cells treated with both systems showed a more pronounced membrane deformations compared to images of cells with each treatment. In conclusion, ultrasound-activated microbubbles can enhance the therapeutic effects of ionizing radiation. In addition, preliminary data from animal experiments indicated that this effect could be reproduced to yield enhanced tumour cell kill in a xenograft model.

Imaging of acute tumor response during cancer therapy may help guide optimization and individualization of treatment regimens. Morphologic changes associated with apoptosis such as cell shrinkage, membrane blebbing and nuclear condensation/fragmentation can change the characteristics of high frequency ultrasound backscatter signal. To monitor tumor response in vivo to radiotherapy and intravascular microbubbles using non-invasive, high frequency ultrasound imaging and spectroscopic analysis. Human prostate cancer xenografts were grown in SCID mice. Treatments consisted of radiotherapy alone (2 Gy or 8 Gy) or in combination with ultrasound activated intravenous microbubbles (low or high concentration). Tumors were assessed before and after therapy using 25 MHz high frequency ultrasound (HFUS) data at 12.5mm focal depth with a 2-3 cm depth of field, using a VisualSonics VS40B scanner, which is capable of collecting and recording raw RF (radiofrequency) data for spectroscopic analysis. A minimum of 5 cross-sectional regions of interest were selected and three spectral parameters were analyzed: 0 MHz-intercept (proportional to scatterer concentration); Spectral Slope (reflecting scatterer size) and Mid-band fit (MBF,) which represents overall backscatter in decibels (dB). Statistical analysis of HFUS spectroscopy employed the paired Student's t test. p values less than 0.05 were considered significant. Eight animals per treatment group (n = 72) were used. Of those, 36 animals were euthanized at the 24 hour time point and tissue specimens used for H&E and TUNEL stains as a histopathological assessment of tumor response. Mid-Band Fit increased in all tumors treated with 8 Gy XRT with no microbubbles, low concentration and high concentration: 4.27 ± 2.15dB (p = 0.005), 2.69 ± 1.66dB (p = 0.048), 5.23 ± 3.44dB (p = 0.014), respectively. With high concentration microbubbles alone and with 2 Gy, the MBF increased 2.37 ± 1.68dB (p = 0.010) and 5.02 ± 1.67dB (p < 0.001), respectively. No significant changes in MBF were seen with 2 Gy radiotherapy alone, low concentration microbubbles alone, or the two combined. 0 MHz-intercept extrapolated from RF data demonstrated similar, statistically significant increases whereas spectral slope was invariant as observed with other cancer treatments. This study demonstrates the ability of HFUS to non-invasively monitor standard and enhanced radiotherapy treatments and may be used to customize treatments in the future.

High-frequency ultrasound has been used in the past to detect apoptosis. It has limited penetration due to attenuation. We have previously demonstrated that deeper penetrating diagnostic conventional-frequency ultrasound can monitor apoptosis with AML-cell samples in vitro. In this study, we evaluated the first-time use of conventional-frequency ultrasound to assess tumor responses to radiation in vivo. Malignant PC3 prostate tumors were treated with a 2 or 8 Gy single fraction of radiation in the presence or absence of endothelial-cell perturbing radiosensitizers (750 kHz activated microbubbles). Tumors were examined by a 10MHz US before and 24h after treatment. Images and backscattered spectroscopic data were acquired. Data were analyzed and compared to standard histopathological markers of apoptotic cell death (including H&E and TUNEL staining). At 24h after radiation, we observed a significant increase in apoptosis histologically with induced radiosensitization. Spectroscopic analysis of ultrasound data indicated increases in spectral slope and mid-band fit (MBF). The MBF increased from -50+/−1 dB in the untreated tumors to -47+/− 1 dB at 24h after exposure to RT alone. In the presence radiosensitization, MBF increase by 5 +/− 1 and 8 +/− 1 dB with 2 and 8 Gy radiation doses, respectively. Such samples upon analysis demonstrated 40% and 54% apoptosis in histopathology analysis whereas at 24h after exposure to radiation alone, only 2-4% apoptosis was detected. The spectral slope in LFUS backscatter was 1.8 dB/MHz prior to treatment that changed to 0.8 and 0.7 dB/MHz with 2 and 8 Gy radiation in the presence of radiation sensitizers respectively. Higher sensitization levels caused more apoptosis in the tumors that could be detected with ultrasound. In conclusion for the first time we demonstrate here, that conventional-frequency ultrasound can detect real-time tumor responses. Assessing such responses early in the course of cancer treatment may provide an opportunity to customize treatments accordingly for individual patients.

The annual International Symposia on Ultrasonic Imaging and Tissue Characterization have long been recognized as the world’s leading forums concerned with ultrasonic techniques for medical diagnosis. This year, sessions will be devoted to Quantitative Ultrasound, Bone, ARFI/Elasticity, Heart, Machine Learning, High-Frequency/Small-Animal Imaging and Imaging. Forty-six technical contributions will be presented at these sessions. A large number of papers will deal with clinical evaluation of novel methodology and instrumentation for tissue characterization.

Microbubbles are commonly used as intravascular contrast agents in diagnostic ultrasound; they can also induce localized biological effects using ultrasound (US) exposure. To investigate ultrasound-activated microbubbles (US-MB) as a novel potential therapeutic enhancer of radiotherapy (XRT) in vivo. Human prostate cancer (PC3) xenografts were grown in SCID mice (n = 72) and exposed to (1) US-MB, 0%, 1% or 3% (v/v) (2) XRT, 0 Gy, 2 Gy or 8 Gy, and (3) all combinations of bubbles and radiation. Microbubbles (Perflutren, Bristol-Myers Squibb) were administered intravenously, activated using 500 kHz US for 70 microseconds over 5 minutes to avoid heating. Radiotherapy consisted of single fraction 160 kVp X-rays. Animal's tissue specimens were assessed using colony assays, histology and immunohistochemistry. Statistical analyses employed the unpaired Student's t tests. US-MB alone caused tumor responses as expected with TUNEL + ve staining per LPF increased to 43 ± 17% vs. 2 ± 2% (p < 0.001) compared with untreated controls. High magnification TUNEL stains confirmed increases in apoptotic cells: 90.4 ± 55 vs. 1 ±1 cells per HPF (p = 0.03). Higher concentration bubbles (3%) significantly increased apoptosis compared with 1% bubbles: 55 ± 11% vs. 31 ± 12% (p = 0.026) area per LPF, and 137 ± 21 vs. 43 ± 22 (p = 0.006) cells per HPF in a primary vascular distribution. US-MB combined with XRT enhanced tumor response compared with XRT alone at all radiation doses. At 2 Gy, percent area per LPF increased: 52 ± 13 vs. 3 ± 2% (p < 0.001) and cell counts per HPF increased: 136 ± 44 vs. 53.5 ± 24 (p = 0.016). For 8 Gy, TUNEL + ve regions increased: 61 ± 9% vs. 1 ± 1% (p < 0.001) per LPF and at high magnification, 148 ± 68 vs. 54 ± 14 (p = 0.029) cells per HPF. TUNEL staining demonstrated apoptosis in diffuse stranding patterns morphologically consistent with pronounced endothelial cell death. The US activated microbubbles can induce enhanced microvascular damage in vivo and enhance tumor response to radiotherapy. We observed a 2 to 4-fold radiosensitization by the combination of therapies which was limited to areas of ultrasound treatment. This represents the first use of this endothelial-cell perturbation method as a novel radiosensitization mechanism. (Nb. The first two authors have made equal contributions to this work.)