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. 2018 Jan 1;8(2):314-327.
doi: 10.7150/thno.19010. eCollection 2018.

Tumour Vascular Shutdown and Cell Death Following Ultrasound-Microbubble Enhanced Radiation Therapy

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Free PMC article

Tumour Vascular Shutdown and Cell Death Following Ultrasound-Microbubble Enhanced Radiation Therapy

Ahmed El Kaffas et al. Theranostics. .
Free PMC article

Abstract

High-dose radiotherapy effects are regulated by acute tumour endothelial cell death followed by rapid tumour cell death instead of canonical DNA break damage. Pre-treatment with ultrasound-stimulated microbubbles (USMB) has enabled higher-dose radiation effects with conventional radiation doses. This study aimed to confirm acute and longitudinal relationships between vascular shutdown and tumour cell death following radiation and USMB in a wild type murine fibrosarcoma model using in vivo imaging. Methods: Tumour xenografts were treated with single radiation doses of 2 or 8 Gy alone, or in combination with low-/high-concentration USMB. Vascular changes and tumour cell death were evaluated at 3, 24 and 72 h following therapy, using high-frequency 3D power Doppler and quantitative ultrasound spectroscopy (QUS) methods, respectively. Staining using in situ end labelling (ISEL) and cluster of differentiation 31 (CD31) of tumour sections were used to assess cell death and vascular distributions, respectively, as gold standard histological methods. Results: Results indicated a decrease in the power Doppler signal of up to 50%, and an increase of more than 5 dBr in cell-death linked QUS parameters at 24 h for tumours treated with combined USMB and radiotherapy. Power Doppler and quantitative ultrasound results were significantly correlated with CD31 and ISEL staining results (p < 0.05), respectively. Moreover, a relationship was found between ultrasound power Doppler and QUS results, as well as between micro-vascular densities (CD31) and the percentage of cell death (ISEL) (R2 0.5-0.9). Conclusions: This study demonstrated, for the first time, the link between acute vascular shutdown and acute tumour cell death using in vivo longitudinal imaging, contributing to the development of theoretical models that incorporate vascular effects in radiation therapy. Overall, this study paves the way for theranostic use of ultrasound in radiation oncology as a diagnostic modality to characterize vascular and tumour response effects simultaneously, as well as a therapeutic modality to complement radiation therapy.

Keywords: power Doppler; quantitative ultrasound spectroscopy; radiation therapy; ultrasound therapy and imaging; ultrasound treatment monitoring; ultrasound-stimulated microbubbles; vascular targeting..

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
(A) Schematic of experimental workflow, imaging time points and treatment conditions. (B) Illustration of events causing tumour cell death where endothelial cells are primary responders/target, followed by tumour cell death.
Figure 2
Figure 2
Top row exhibits volumetric maximum intensity projections of tumour power Doppler signal for a control tumour at 24 h, as well as following 2 Gy alone, or in combination with Low USMB and High USMB (all at 24 h). Results qualitatively demonstrate a decrease in power Doppler signal as a function of treatment. The second row exhibits 2D B-mode images of a single plain in the center of a tumour volume with a rectangular overlay as a representative ROI used for QUS analysis. Images are of post-treated tumours. The third row exhibits parametric map overlays of the MBF in the selected ROIs. One can observe an increase in signal as a function of treatment. Color legend bars encompass 40 dB for power Doppler and 12 dBr for the MBF parametric map images. Yellow arrows indicate the tumour tissue-skin boundary. Scale bars denote 2.2 mm for power Doppler and 1 mm for Bmode/QUS parametric map.
Figure 3
Figure 3
(A) Quantified power Doppler vascularity index (VI) at 3 h, 24 h and 72 h for control, single doses of 2 Gy or 8 Gy radiation, low and high concentration of USMB, and combined treatment permutations. The graphs exhibit the relative change of Doppler signal represented as the relative VI. There is a decrease in the VI for combined microbubble and radiation treated tumours when compared with the radiation only conditions, entailing an enhanced radiotherapy effect with the use of microbubbles. (B) Quantified MBF for the same treatment conditions. Similar to power Doppler results, we note a greater increase in the MBF QUS parameter in animals treated with radiation and microbubbles, in comparison to those treated with radiation or microbubbles alone. Significance is indicated by * for p < 0.05. All conditions are compared to the control condition (0 Gy and Nil microbubbles of the corresponding time points).
Figure 4
Figure 4
(A) Representative images of ISEL (top) and CD31 (bottom) stained tumour cross-sections obtained at 24 h post-treatment. Panel demonstrates qualitative increases in cell death and decreases in the microvasculature density in treatment when comparing the control and treated conditions. The scale bars denote 1mm and 0.2 mm in ISEL and CD31, respectively. (B) Quantified ISEL stained tumour cross-sections as a gold standard measurement of tumour cell death. We note an overall increase in cell death in animals treated with the combination treatments. (C) Quantified CD31 stained tumour cross sections, expressed as the MVD. Results confirm a decrease in vascularity in animals receiving combined therapy. Significance is indicated by * for p < 0.05. All conditions are compared to the control condition (0 Gy and Nil microbubbles of the corresponding time points).
Figure 5
Figure 5
(A) Correlations between quantified CD31 results and the power Doppler VI parameter. Good correlations are noted at 24 h and 72 h. (B) Correlations between quantified ISEL results and the QUS-MBF parameter. A good correlative agreement is noted, especially at 24 h and 72 h. These confirm ultrasound-based link to gold standard histology.
Figure 6
Figure 6
(A) First-order correlations between the VI and the MBF. (B) Correlations between the percent cell death (ISEL) and the MVD (CD31). Correlations with an R2 > 0.7 are found at 24 h.

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