ABSTRACT
Non-invasive monitoring of cancer cell death would permit rapid feedback on treatment response. One technique showing such promise is quantitative ultrasound. High-frequency ultrasound spectral radiofrequency analysis was used to study cell death in breast cancer cell samples. Quantitative ultrasound parameters, including attenuation, spectral slope, spectral 0-MHz-intercept, midband fit, and fitted parameters displayed significant changes with paclitaxel-induced cell death, corresponding to observations of morphological changes seen in histology and electron microscopy. In particular, a decrease in spectral slope from 0.24±0.07 dB/MHz to 0.04±0.09 dB/MHz occurred over 24 hours of treatment time and was identified as an ultrasound parameter capable of differentiating post-mitotic arrest cell death from classical apoptosis. The formation of condensed chromatin aggregates of 1 micron or greater in size increased the number of intracellular scatterers, consistent with a hypothesis that nuclear material is a primary source of ultrasound scattering in dying cells. It was demonstrated that the midband fit quantitatively correlated to cell death index, with a Pearson R-squared value of 0.99 at p<0.01. These results suggest that high-frequency ultrasound can not only qualitatively assess the degree of cancer cell death, but may be used to quantify the efficacy of chemotherapeutic treatments.
ABSTRACT
High frequency quantitative ultrasound techniques were investigated to characterize different forms of cell death in vitro. Suspension-grown acute myeloid leukemia cells were treated to cause apoptosis, oncosis, mitotic arrest, and heat-induced death. Samples were scanned with 20 and 40 MHz ultrasound and assessed histologically in terms of cellular structure. Frequency-domain analysis of 20 MHz ultrasound data demonstrated midband fit changes of 6.0 ± 0.7 dBr, 6.2 ± 1.8 dBr, 4.0 ± 1.0 dBr and -4.6 ± 1.7 dBr after 48-hour cisplatinum-induced apoptosis, 48-hour oncotic decay, 36-hour colchicine-induced mitotic arrest, and heat treatment compared to control, respectively. Trends from 40 MHz ultrasound were similar. Spectral slope changes obtained from 40 MHz ultrasound data were reflective of alterations in cell and nucleus size. Chromatin pyknosis or lysis trends suggested that the density of nuclear material may be responsible for observed changes in ultrasound backscatter. Flow cytometry analysis confirmed the modes of cell death and supported midband fit trends in ultrasound data. Scatterer-size and concentration estimates obtained from a fluid-filled sphere form factor model further corresponded with spectral analysis and histology. Results indicate quantitative ultrasound spectral analysis may be used for probing anti-cancer response and distinguishing various modes of cell death in vitro.
ABSTRACT
Current methods to evaluate the status of a cell are largely focused on fluorescent identification of molecular biomarkers. The invasive nature of these methods - requiring either fixation, chemical dyes, genetic alteration, or a combination of these - prevents subsequent analysis of samples. In light of this limitation, studies have considered the use of physical markers to differentiate cell stages. Acoustic microscopy is an ultrahigh frequency (>100 MHz) ultrasound technology that can be used to calculate the mechanical and physical properties of biological cells in real-time, thereby evaluating cell stage in live cells without invasive biomarker evaluation. Using acoustic microscopy, MCF-7 human breast adenocarcinoma cells within the G1, G2, and metaphase phases of the proliferative cell cycle, in addition to early and late programmed cell death, were examined. Physical properties calculated include the cell height, sound speed, acoustic impedance, cell density, adiabatic bulk modulus, and the ultrasonic attenuation. A total of 290 cells were measured, 58 from each cell phase, assessed using fluorescent and phase contrast microscopy. Cells actively progressing from G1 to metaphase were marked by a 28% decrease in attenuation, in contrast to the induction of apoptosis from G1, which was marked by a significant 81% increase in attenuation. Furthermore late apoptotic cells separated into 2 distinct groups based on ultrasound attenuation, suggesting that presently-unidentified sub-stages may exist within late apoptosis. A methodology has been implemented for the identification of cell stages without the use of chemical dyes, fixation, or genetic manipulation.
