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. 2018 Sep 1;110(9):1009-1018.
doi: 10.1093/jnci/djy011.

Role of Acid Sphingomyelinase and Ceramide in Mechano-Acoustic Enhancement of Tumor Radiation Responses

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Free PMC article

Role of Acid Sphingomyelinase and Ceramide in Mechano-Acoustic Enhancement of Tumor Radiation Responses

Ahmed El Kaffas et al. J Natl Cancer Inst. .
Free PMC article

Abstract

Background: High-dose radiotherapy (>8-10 Gy) causes rapid endothelial cell death via acid sphingomyelinase (ASMase)-induced ceramide production, resulting in biologically significant enhancement of tumor responses. To further augment or solicit similar effects at low radiation doses, we used genetic and chemical approaches to evaluate mechano-acoustic activation of the ASMase-ceramide pathway by ultrasound-stimulated microbubbles (USMB).

Methods: Experiments were carried out in wild-type and acid sphingomyelinase (asmase) knockout mice implanted with fibrosarcoma xenografts. A cohort of wild-type mice received the ASMase-ceramide pathway inhibitor sphingosine-1-phosphate (S1P). Mice were treated with varying radiation doses, with or without a priori USMB exposure at different microbubble concentrations. Treatment response was assessed with quantitative 3D Doppler ultrasound and immunohistochemistry at baseline, and at three, 24, and 72 hours after treatment, with three to five mice per treatment group at each time point. All statistical tests were two-sided.

Results: Results confirmed an interaction between USMB and ionizing radiation at 24 hours (P < .001), with a decrease in tumor perfusion of up to 46.5% by three hours following radiation and USMB. This peaked at 24 hours, persisting for up to 72 hours, and was accompanied by extensive tumor cell death. In contrast, statistically nonsignificant and minimal tumor responses were noted in S1P-treated and asmase knockout mice for all treatments.

Conclusions: This work is the first to confirm the involvement of the ASMase-ceramide pathway in mechanotransductive vascular targeting using USMB. Results also confirm that an acute vascular effect is driving this form of enhanced radiation response, and that it can be elicited at low radiation doses (<8-10 Gy) by a priori USMB exposure.

Figures

Figure 1.
Figure 1.
Overview of hypothesis and method schematics. A) Hypothesized biological mechanism behind ultrasound-stimulated microbubbles (USMB) and radiation treatments involving acid sphingomyelinase (ASMase) and ceramide. Whereas high doses (>8 Gy) of radiation alone activate sufficient ceramide to message for endothelial cell death, radiation doses lower than 2–6 Gy do not release sufficient quantities to activate ceramide-induced cell death. Similarly, whereas ceramide is released following USMB treatments, the amount is not sufficient to activate the rapid and extensive endothelial cell death needed for vascular shutdown. In contrast, combining radiation (low or high dose) with USMB releases sufficient ceramide to surpass the threshold, resulting in extensive tumor endothelial cell death and vascular shutdown. Because the microbubbles used have an average diameter of 3 μm and thus remain within blood vessels, treatment with USMB mechanically targets endothelial cells that surround the flowing intravascular microbubbles. B) Schematic of experimental conditions and imaging and treatment workflow. C) Hypothesized therapeutic mechanism following USMB and radiation treatments. In tissues, we posit that the combined USMB and radiation treatment causes vascular perturbation, a disruption in blood flow, and changes in oxygenation, leading to cell death, consistent with what appears to be potentially ischemic tumor cell death. The rationale behind the approach is that areas with induced anoxic cell death do not require any further therapeutic doses of radiation due to the massive vascular destruction already caused by an initial USMB and radiation treatment (12,32,33). D) Schematic of treatment pulse used to stimulate microbubbles (total USMB treatment time was five minutes, with 16-cycle tone burst, 3 kHz pulse repetition frequency, duty cycle of 10%, peak negative acoustic pressure of 500 kPa, mechanical index of 0.8). ko = knockout; MB = microbubble; S1P = sphingosine-1-phosphate; USMB = ultrasound-stimulated microbubble; wt = wild-type; XRT = ionizing radiation.
Figure 2.
Figure 2.
Power Doppler results. A and B) Tumor results of maximum intensity projections of the power Doppler signal in volumetric images for various treatment conditions. The scale bar is 2 mm, and the color bar is 0 (black) to 40 dB (yellow). A and B) Results 24 hours after treatment with radiation alone, and radiation in combination with ultrasound-stimulated microbubble (USMB) exposure, respectively. C and D) Quantified relative vascularity index at three hours, 24 hours, and 72 hours for each of the radiation and USMB (low is 1%, and high is 3% volume of total blood volume [v/v]) treatment conditions in wild-type (wt) and sphingosine-1-phosphate (S1P)-treated mice. The three- and 72-hour time points have four mice per treatment; the 24-hour time has five mice per treatment. Pairwise multiple-comparisons were performed using two-sided Tukey’s Honest Significant Difference Procedure to test for statistical significance following analysis of variance. All P values are displayed for statistically significant treatment conditions in the graph. All statistical comparisons are compared with the 0 Gy, 0% ultrasound-stimulated microbubbles (Nil) condition at the same time and are summarized in Supplementary Tables 1 and 2 (available online). Error bars represent SD. S1P = sphingosine-1-phosphate; wt = wild-type; VI = vascularity index.
Figure 3.
Figure 3.
Cluster of differentiation 31 (CD31) results. A and B) Representative images of CD31-stained tumor sections from wild-type (wt) and sphingosine-1-phosphate (S1P)-treated mice following radiation and ultrasound-stimulated microbubble (USMB; low is 1%, and high is 3% volume of total blood volume [v/v]). The scale bar represents 50 μm. C and D) Quantified CD31 staining for all radiation doses and time points in wild-type (wt) and S1P-treated mice. The following wt treatment conditions have three mice each: all 3% USMB at three hours; all 1% or 3% USMB at 72 hours. All other wt treatment conditions have four mice each. The following S1P treatment conditions have three mice each: all 1% USMB at three hours; 3% USMB with 2 Gy at three hours; 2 Gy only; 1% USMB with 0 Gy and 2 Gy at 24 hours; control at 72 hours; 1% USMB with 2 Gy at 72 hours. All other S1P treatment conditions have four mice each. Pairwise multiple comparisons were performed using two-sided Tukey’s Honest Significant Difference Procedure to test for statistical significance following analysis of variance. All P values are displayed for statistically significant treatment conditions. All statistical comparisons are compared with the 0 Gy, 0% ultrasound-stimulated microbubbles (Nil) condition at the same time and are summarized in Supplementary Tables 1 and 2 (available online). Error bars represent SD. MVD = microvascular density; S1P = sphingosine-1-phosphate; wt = wild-type.
Figure 4.
Figure 4.
Tumor cell death results. A and B) Representative images of in situ end labeling (ISEL)–stained tumor cross-sections from wild-type (wt) and sphingosine-1-phosphate (S1P)-treated mice following ultrasound-stimulated microbubbles (USMB; low is 1%, and high is 3% volume of total blood volume [v/v]) and radiation. The scale bar represents 1 mm. C and D) Quantified ISEL staining for all radiation doses and time points in wild-type and S1P-treated mice. All wt treatment conditions include four mice each, except for radiation-only conditions at 72 hours. All S1P treatment conditions include four mice each. Pairwise multiple comparisons were performed using two-sided Tukey’s Honest Significant Difference Procedure to test for statistical significance following analysis of variance. All P values are displayed for statistically significant treatment conditions. Statistical comparisons are compared with the 0 Gy, 0% USMB (Nil) condition at the same time point and are summarized in Supplementary Tables 1 and 2 (available online). Error bars represent SD. ISEL = in situ end labeling; S1P = sphingosine-1-phosphate; wt = wild-type.
Figure 5.
Figure 5.
Power Doppler and histological results for wild-type (wt) and asmase knockout mice at 24 hours after irradiation. A) Representative results of maximum intensity projections of the power Doppler signal in volumetric images for various treatment conditions. The scale bar is 2 mm, and the color bar is 0 (black) to 40 dB (yellow). The scale bar represents 1 mm. B) Representative images of cluster of differentiation (CD31)–stained tumor sections from wild-type and knockout mice following radiation and ultrasound-stimulated microbubbles (USMB; low is 1%, and high is 3% volume of total blood volume [v/v]). The scale bar is 60 μm. C) Representative images of in situ end labeling (ISEL)–stained tumor cross-sections from wild-type (wt) and knockout (ko) mice following USMB and radiation. The scale bar is 1.3 mm. D–F) Quantified power Doppler, CD31, and ISEL results for wild-type and knockout mice. All ko treatment conditions in (D–F) have five mice each. Pairwise multiple comparisons were performed using two-sided Tukey’s Honest Significant Difference Procedure to test for statistical significance following analysis of variance. All P values are displayed for statistically significant treatment conditions. All statistical comparisons are compared with the 0 Gy, 0% USMB (Nil) condition at the same time point and are summarized in Supplementary Table 3 (available online). Error bars represent SD. ISEL = in situ end labeling; MVD = microvascular density; VI = vascularity index.
Figure 6.
Figure 6.
Ceramide staining and tumor growth in wild-type and S1P-treated mice. A) Tumor sections triple labeled for ceramide (red), endothelial cells (cluster of differentiation [CD31]; green), and cellular nuclei (double stranded DNA [DAPI]; blue). Ceramide (red fluorescence) is elevated in treated wild-type (wt) mice; minimal ceramide is noted in sphingosine-1-phosphate (S1P) and knockout (ko) mice. Arrows point to the labeling of endothelial cells lining blood vessels (CD31 labeling, green fluorescence) that appear yellowish-orange in treated wt due to the overlay of the red (ceramide) and the green (CD31); DAPI is a nuclear stain (blue fluorescence). The scale bar is 20 μm. B) Quantified ceramide for wild-type, S1P, and knockout mice. Each treatment condition has four mice. Error bars represent SEM. C) Tumor growth normalized to baseline for wild-type and S1P mice treated with high ultrasound-stimulated microbubbles (USMB; high is 3% volume of total blood volume [v/v]) and ionizing radiation (8 Gy). For wt growth curves, there are 10 mice in the control group, eight mice in the 8 Gy group, 10 mice in 3% USMB with 0 Gy, and 12 mice in 3% USMB with 8 Gy. For S1P growth curves, there are five mice in the control group, six mice in the 8 Gy group, five mice in 3% USMB with 0 Gy, and five mice in 3% USMB with 8 Gy. All statistical comparisons are carried out with an unpaired two-tailed t test and compared with the 0 Gy, 0% USMB (Nil) condition at the same time point. Samples from four tumors per condition were labeled. Error bars represent standard deviations. Tumor volume doubling time was approximately five to six, 12, eight, and >20 days for control, 8 Gy, 0 Gy with high USMB, and 8 Gy with high USMB treatments, respectively. In contrast, S1P-treated mice experienced minimal growth delay with an approximate tumor doubling time of four, three, eight, and eight days for control, 8 Gy, 0 Gy with high USMB, and 8 Gy with high USMB, respectively. Error bars represent SD. ko = knockout; S1P = sphingosine-1-phosphate; wt = wild-type.

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