Quantitative Ultrasound Characterization of Tumor Cell Death: Ultrasound-Stimulated Microbubbles for Radiation Enhancement

Abstract

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.

Ultrasound Microbubble Treatment Enhances Clathrin-Mediated Endocytosis and Fluid-Phase Uptake through Distinct Mechanisms

“…Indeed we have previously reported that acute (90 min) activation of AMP-activated protein kinase (AMPK), mimicking conditions that occur during metabolic stress, results in significant changes to the cell surface proteome [ 53 ], largely as a result of changes in membrane traffic. USMB is being combined with clinical cancer treatment methods such as radiotherapy and chemotherapy as a means of enhancing treatment effectiveness and safety [ 9 , 10 , 54 – 56 ]. Hence, the broad regulation of CME by USMB treatment may also effect large, systematic reprogramming of the cell surface proteome that may have implications for the use of USMB in combination therapy to treat cancer.…”

Quantitative ultrasound imaging of therapy response in bladder cancer in vivo

“…Another study by Kim et al . showed the relationship between cell fragmentation and QUS; demonstrating a 17-fold increase in cell fragmentation relative to a change in the mid-band fit and 0-MHz Intercept (7.0 ± 4.1 dBr and 15.4 ± 2.5 dBr, respectively) [ 7 ]. In photodynamic-treated (PDT) melanoma treatments, Banihashemi and colleagues used high frequency ultrasound (26 MHz) to detect apoptotic cell death, and showed a statistically significant correlation between QUS changes and cell death up to 24 hours after PDT treatment [ 10 ].…”

“…However, at higher acoustic pressures, ultrasound can cause microbubble disruption through cavitation and oscillations. Previous studies have shown that when bubbles are introduced to the tumor vasculature and insonified using high mechanical indices, the mechanical forces can perturb endothelial cells within the vessels themselves [ 7 ]. There is evidence to support that this disruption initiates cell death signalling similar to radiation response mechanisms, and can lead to additive tumor damage [ 8 ].…”

“…Structural alterations such as, pyknosis and karyorhexis caused by chemotherapyinduced cell death were observed following treatment and showed an increase in the backscatter intensity, as early as 24 hours after treatment [ 19 , 20 ]. These QUS techniques have since been demonstrated to be effective in monitoring a variety of tumor models including skin, head and neck, prostate, breast, and acute myeloid leukemia [ 7 , 10 , 11 , 21 - 23 ]. In the present study, we apply QUS techniques to investigate treatment-induced cell death in bladder cancer xenografts and build on our previous report using ultrasound-stimulated microbubbles and radiation in vivo [ 5 ].…”

Ultrasound-stimulated microbubble radiation enhancement of tumors: Single-dose and fractionated treatment evaluation

“…Furthermore, acute vascular shutdown and significant tumor cell death is observed [33,34], indicating that tumor cell death is secondary to endothelial cell death. Several in vivo studies have also indicated the effectiveness of this therapy in treating xenograft tumors in murine models, including breast, prostate liver, and bladder [21,22,[35][36][37][38][39][40].…”