Gregory J. Czarnota

Ultrasound-Activated Microbubble Cancer Therapy: Ceramide Production Leading to Enhanced Radiation Effect in vitro

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.

Breast tumor response to ultrasound mediated excitation of microbubbles and radiation therapy in vivo

Acoustically stimulated microbubbles have been demonstrated to perturb endothelial cells of the vasculature resulting in biological effects. In the present study, vascular and tumor response to ultrasound-stimulated microbubble and radiation treatment was investigated in vivo to identify effects on the blood vessel endothelium. Mice bearing breast cancer tumors (MDA-MB-231) were exposed to ultrasound after intravenous injection of microbubbles at different concentrations, and radiation at different doses (0, 2, and 8 Gy). Mice were sacrificed 12 and 24 hours after treatment for histopathological analysis. Tumor growth delay was assessed for up to 28 days after treatment. The results demonstrated additive antitumor and antivascular effects when ultrasound stimulated microbubbles were combined with radiation. Results indicated tumor cell apoptosis, vascular leakage, a decrease in tumor vasculature, a delay in tumor growth and an overall tumor disruption. When coupled with radiation, ultrasound-stimulated microbubbles elicited synergistic anti-tumor and antivascular effects by acting as a radioenhancing agent in breast tumor blood vessels. The present study demonstrates ultrasound driven microbubbles as a novel form of targeted antiangiogenic therapy in a breast cancer xenograft model that can potentiate additive effects to radiation in vivo.

Quantitative ultrasound imaging of therapy response in bladder cancer in vivo

Background and Aims Quantitative ultrasound (QUS) was investigated to monitor bladder cancer treatment response in vivo and to evaluate tumor cell death from combined treatments using ultrasound-stimulated microbubbles and radiation therapy. Methods Tumor-bearing mice (n=45), with bladder cancer xenografts (HT- 1376) were exposed to 9 treatment conditions consisting of variable concentrations of ultrasound-stimulated Definity microbubbles [nil, low (1%), high (3%)], combined with single fractionated doses of radiation (0 Gy, 2 Gy, 8 Gy). High frequency (25 MHz) ultrasound was used to collect the raw radiofrequency (RF) data of the backscatter signal from tumors prior to, and 24 hours after treatment in order to obtain QUS parameters. The calculated QUS spectral parameters included the mid-band fit (MBF), and 0-MHz intercept (SI) using a linear regression analysis of the normalized power spectrum. Results and Conclusions There were maximal increases in QUS parameters following treatments with high concentration microbubbles combined with 8 Gy radiation: (ΔMBF = +6.41 ± 1.40 (±SD) dBr and SI= + 7.01 ± 1.20 (±SD) dBr. Histological data revealed increased cell death, and a reduction in nuclear size with treatments, which was mirrored by changes in quantitative ultrasound parameters. QUS demonstrated markers to detect treatment effects in bladder tumors in vivo.

Implications of tumor oxygenation and blood flow for cancer treatment monitoring using photoacoustic imaging and power Doppler

Photoacoustic (PA) imaging has been proposed for cancer treatment monitoring. Tumor oxygen saturation (sO2) should in principle be related to vascular parameters such as blood flow. In this work, in-vivo PA estimates of sO2 were compared to power Doppler (pD) measures of vascularity hours after the administration of microbubbles (MB), radiation therapy (XRT), individually or combined (MB-XRT).

Abstract 1812: Ultrasound-stimulated microbubble based biomechanical enhancement of radiation cell death: Role of acid sphingomyelinase and ceramide

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAnnWe 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.nnExperiments 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.nnResults 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 < 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.nnCitation 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

P6C-4 Extended System Transfer Compensation for Parametric Imaging in Ultrasonic Response Assessment of Anti-Cancer Therapies

The assessment of the tissue response in anti cancer therapy is a time critical process. The early recognition of a failing treatment might allow an adjustment to increase the success rate and spare unnecessary side effects. Today biopsy and nuclear medicine are commonly used procedures for assessing the treatment success. However biopsies are invasive and provide a limited sample volume and nuclear medicine on the other hand requires the application of radioactive agents. It has been observed that ultrasound backscatter properties of cell collections are altered when the cells are respond to an oncological treatment. In previous studies we have estimated spectral properties of ultrasound backscatter using commonly accepted procedures for eliminating system specific transfer properties. These methods proved to be sufficient when investigating regions close to the transducers focus. However, the application in a clinical environment will require the parameter estimation in an extended area combined with parametric imaging. The purpose of this work is to implement more accurate methods for the determination of the effective scatterer size in quantitative high frequency ultrasound. To improve the accuracy but also extend the axial image depth for parametric imaging we developed an algorithm for eliminating the transfer properties of the equipment. It accounts for the distortion of the incident pulse due to the relative defocus position of a time gate and the corresponding alterations in the spectral shape. Two different methods for estimating the transfer properties were applied. One method used the echoes obtained from a plane reflector at 51 positions within the plusmn 5 mm range around the transducers focus. The second method derived the required compensation function from the signal variation within a pellet of untreated cells. Both, the axial amplitude variation and the alterations of the spectral shape were derived as a function of the defocus position. The slope of the normalized power spectrum, the effective scatterer size and integrated backscatter coefficients were computed from ultrasound backscatter of cervix carcinoma (HeLa) cells after applying the compensation algorithms. Chemotherapy was applied to induce apoptosis in HeLa cells. At 6 time points after treatment cells were harvested and ultrasound backscatter was recorded using a 20 MHz (f# 2.35) and a 40 MHz (f# 3) transducer. Within the axial -12 dB range of the transducer slope differences of up to -1.2 dB/MHz were observed and compensated. Integrated backscatter coefficients increased by over 300% of the initial values. This study contributes towards a non-invasive method for estimating tissue responses in anti-cancer therapy.