Abstract 1812: Ultrasound-stimulated microbubble based biomechanical enhancement of radiation cell death: Role of acid sphingomyelinase and ceramide
Authors: Czarnota Gregory Jan , El Kaffas Ahmed , Giles Anoja , Al Mahrouki Azza , Hashim Amr 
Published: August 2015

We have recently demonstrated that mechanical perturbation of endothelial cells from ultrasound-stimulated microbubbles (USMB) results in enhanced tumour radiosensitivity. When exposed to high-power ultrasound, microbubbles release energy in the form of local mechanical forces on the membranes of surrounding endothelial cells. Upon irradiation, rapid vascular shutdown occurs followed by extensive secondary tumour cell death. Our hypothesis is that USMB-based endothelial membrane perturbations produce ceramide via a sphingomyelinase (ASMase) pathway, and act synergistically with radiation to enhance overall tumour response. Here we investigate the role of the SMase-ceramide pathway on USMB-based endothelial radiosensitization.

Experiments were carried out in wild type (C57BL/6) and ASMase knockout mice, implanted with a fibrosarcoma line (MCA-129). Animals were treated with radiation doses varying from 0-8 Gy alone, or in combination with USMB. In addition, we pre-treated a subset of animals with sphingosine-1-phosphate (S1P). Treatments with USMB consisted of a 16-cycle tone burst at a 500 kHz center frequency and 570 kPa using a 2.86 cm element diameter ultrasound transducer. The total insonification time was 750 ms over 5 minutes. Microbubbles (Lantheus Medical) were injected through the tail-vein resulting in a blood volume concentration of 1% or 3% (v/v). Treatment response was assessed with Doppler ultrasound acquired at 3, 24 and 72 hrs using a VEVO770 preclinical ultrasound system. The vascularity index (VI) was used to quantify power Doppler data. Staining using ISEL, ceramide and CD31 immunohistochemistry of tumour sections was used to complement ultrasound images, and to confirm results.

Results suggest a link between USMB-based radiosensitization and ASMase. We observed an average decrease of 30% in the VI by 3 hrs in wild type tumours receiving 8 Gy radiation alone. In contrast, those receiving 8 Gy and USMB resulted in a VI decrease of up to 50%. Similarly, while 2 Gy alone induced minimal effects on the tumour vasculature, combining it with USMB resulted in up to 40% decrease in the VI by 24 hrs. Vascular effects were significant (p &lt; 0.05) and sustained for up to 72 hrs after combined treatments. These resulted in significant cell death. In contrast to wild type animals, ASMase knockout mice, or wild-type mice receiving S1P, were found to be generally resistant to the anti-vascular effects of radiation and USMB. We noted minimal cell death and no vascular shutdown following any of the treatments in those experimental groups. Overall conclusions drawn from this work point to a mechanotransduction-like effect that results in endothelial radiosensitization.

Citation Format: Gregory Jan Czarnota, Ahmed El Kaffas, Anoja Giles, Azza Al Mahrouki, Amr Hashim. Ultrasound-stimulated microbubble based biomechanical enhancement of radiation cell death: Role of acid sphingomyelinase and ceramide. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1812. doi:10.1158/1538-7445.AM2015-1812