Quantitative ultrasound characterization of tumor cell death: ultrasound-stimulated microbubbles for radiation enhancement.

The aim of this study was to assess the efficacy of quantitative ultrasound imaging in characterizing cancer cell death caused by enhanced radiation treatments. This investigation focused on developing this ultrasound modality as an imaging-based non-invasive method that can be used to monitor therapeutic ultrasound and radiation effects. High-frequency (25 MHz) ultrasound was used to image tumor responses caused by ultrasound-stimulated microbubbles in combination with radiation. Human prostate xenografts grown in severe combined immunodeficiency (SCID) mice were treated using 8, 80, or 1000 µL/kg of microbubbles stimulated with ultrasound at 250, 570, or 750 kPa, and exposed to 0, 2, or 8 Gy of radiation. Tumors were imaged prior to treatment and 24 hours after treatment. Spectral analysis of images acquired from treated tumors revealed overall increases in ultrasound backscatter intensity and the spectral intercept parameter. The increase in backscatter intensity compared to the control ranged from 1.9±1.6 dB for the clinical imaging dose of microbubbles (8 µL/kg, 250 kPa, 2 Gy) to 7.0±4.1 dB for the most extreme treatment condition (1000 µL/kg, 750 kPa, 8 Gy). In parallel, in situ end-labelling (ISEL) staining, ceramide, and cyclophilin A staining demonstrated increases in cell death due to DNA fragmentation, ceramide-mediated apoptosis, and release of cyclophilin A as a result of cell membrane permeabilization, respectively. Quantitative ultrasound results indicated changes that paralleled increases in cell death observed from histology analyses supporting its use for non-invasive monitoring of cancer treatment outcomes

Evaluating the effects of radiation and acoustically-stimulated microbubble therapy in an in vivo breast cancer model.

Ultrasound-stimulated microbubbles (USMB) cause localized vascular effects and sensitize tumors to radiation therapy (XRT). We investigated acoustic parameter optimization for combining USMB and XRT. We treated breast cancer xenograft tumors with 500 kHz pulsed ultrasound at varying pressures (570 or 740 kPa), durations (1 to 10 minutes), and microbubble concentrations (0.01 to 1% (v/v)). Radiation therapy (2 Gy) was administered immediately or after a 6-hour delay. Histological staining of tumors 24 hours after treatment detected changes in cell morphology, cell death, and microvascular density. Significant cell death resulted at 570 kPa after a 1-minute exposure with 1% (v/v) microbubbles with or without XRT. However, significant microvascular disruption required higher ultrasound pressure and exposure duration greater than 5 minutes. Introducing a 6-hour delay between treatments (USMB and XRT) showed a similar tumor effect with no further improvement in response as compared to when XRT was delivered immediately after USMB

High frequency ultrasound B-mode images with ROI parametric overlays of the mid-band fit biomarker for PC-3 xenografts.

<p>(A) Tumors treated with varying radiation doses (0–8 Gy) without ultrasound-microbubble treatment. (B) Tumors treated with varying microbubble concentrations (8–1000 µL/kg) combined with 750 kPa of ultrasound pulse and 8 Gy-radiation. (C) Tumors treated with varying ultrasound pressures (250–750 kPa) combined with 8 µL/kg of microbubbles and 8 Gy-radiation. The scale bar represents 4 mm.</p

High magnification light microscope images of H&E-stained PC-3 xenografts.

<p>In addition to control, tumors treated with ultrasound pulses at 750(A) The panel shows H&E stained cells that exhibit condensed and/or fragmented apoptotic bodies. Each Panel demonstrates a representative region of cell death within tumor. Microbubble concentrations are given as 8 µL/kg, 80 µL/kg, and high 1000 µL/kg. The scale bar represents 25 µm. (B) Quantification of normalized fraction of condensed or fragmented nuclei. Each graph shows the fraction of cell death and disruption for varied microbubble concentration and radiation doses at a fixed ultrasound peak-negative pressure: 250 kPa, 570 kPa, and 750 kPa. Each bar represents the mean value of five samples (n = 5) and the error bar indicates the standard error. Statistical testing using 2-way ANOVA indicates the effects caused by the changes in both microbubble concentration and dose of radiation to be very significant (<i>p</i><0.0001).</p

Results of statistical analysis performed on the “<i>Quantification of normalized fraction of condensed or fragmented nuclei</i>” using one-tailed paired t-test.

<p>Results of statistical analysis performed on the “<i>Quantification of normalized fraction of condensed or fragmented nuclei</i>” using one-tailed paired t-test.</p

Average changes in midband-fit parameter.

<p>Each bar represents the mean of midband-fit values of five mouse-borne tumors (n = 5). The error bar indicates the standard error within the sample size. Statistical testing using 2-way ANOVA indicates the effects caused by the changes in both microbubble concentration and dose of radiation to be very significant for every graph (<i>p</i><0.0001). Each graph shows the average changes in midband-fit for varied microbubble concentration and radiation doses at a fixed ultrasound pressure: 250 kPa, 570 kPa, and 750 kPa.</p

Results of statistical analysis performed on the “Quantification of normalized fraction of condensed or fragmented nuclei” using 2-way ANOVA without replication.

<p>Results of statistical analysis performed on the “Quantification of normalized fraction of condensed or fragmented nuclei” using 2-way ANOVA without replication.</p

High-frequency ultrasound B-mode images of PC-3 xenografts.

<p>B-Mode (left) and on the right side of each row, respective representative normalized power spectra are shown. (A) Tumors treated with varying radiation doses (0–8 Gy) without ultrasound-microbubble treatment. (B) Tumors treated with varying microbubble concentrations (8–1000 µL/kg) combined with 750 kPa of ultrasound pulse and 8 Gy-radiation. (C) Tumors treated with varying ultrasound pressures (250–750 kPa) combined with 8 µL/kg of microbubbles and 8 Gy-radiation. The scale bar represents 2 mm.</p

Results of statistical analysis performed on the “<i>Average Changes in Midband-fit, 0-MHz Intercept, and Slope parameter</i>” using 2-way ANOVA without replication.

<p>Results of statistical analysis performed on the “<i>Average Changes in Midband-fit, 0-MHz Intercept, and Slope parameter</i>” using 2-way ANOVA without replication.</p

Results of statistical analysis performed on the “Quantification of Ceramide” using 2-way ANOVA without replication.

<p>Results of statistical analysis performed on the “Quantification of Ceramide” using 2-way ANOVA without replication.</p