Quantitative evaluation of cell death response in vitro and in vivo using conventional-frequency ultrasound

Abstract

Previous studies using high-frequency ultrasound have suggested that radiofrequency (RF) spectral analysis can be used to quantify changes in cell morphology to detect cell death response to therapy non-invasively. The study here investigated this at conventional-frequencies, frequently used in clinical settings. Spectral analysis was performed using ultrasound RF data collected with a clinical ultrasound platform. Acute myeloid leukemia (AML-5) cells were exposed to cisplatinum for 0-72 hours in vitro and prepared for ultrasound data collection. Preclinical in vivo experiments were also performed on AML-5 tumour-bearing mice receiving chemotherapy. The mid-band fit (MBF) spectral parameter demonstrated an increase of 4.4 ± 1.5 dBr for in vitro samples assessed 48 hours after treatment, a statistically significant change (p < 0.05) compared to control. Further, in vitro concentration-based analysis of a mixture of apoptotic and untreated cells indicated a mean change of 10.9 ± 2.4 dBr in MBF between 0% and 40% apoptotic cell mixtures. Similar effects were reproduced in vivo with an increase of 4.6 ± 0.3 dBr in MBF compared to control, for tumours with considerable apoptotic areas within histological samples. The alterations in the size of cells and nuclei corresponded well with changes measured in the quantitative ultrasound (QUS) parameters.

Keywords: apoptosis; cancer therapy; personalized medicine; quantitative ultrasound; treatment response monitoring.

Figure 1. Representative ultrasound data acquired in vitro

A. Conventional-frequency ultrasound B-mode images obtained from control and treated AML-5 cell samples. Rectangles demonstrate the ROIs for the spectral analysis. The scale bar represents ~ 1 mm. B. Normalized power spectra illustrating the typical disparity in intensity and shape of ultrasound backscatter power spectra collected from control and treated cell samples.

Figure 2. Representative light microscopy images of histology slides at 40× magnification from in vitro experiments

A. H&E stained slides and B. TUNEL stained slides obtained from control and treated AML-5 cell samples. The scale bars in (A) and (B) represent ~ 20 μm and ~ 50 μm, respectively.

Figure 3. Results of time-dependent histological analysis on AML-5 cell samples in vitro

A. Representative light microscopy images of H&E stained slides at 40× magnification, corresponding to 0, 6, 12, 24, 48 and 72 hours after the treatment. The scale bar represents ~ 20 μm. B. Average sizes of the cells and their nuclei measured before and at different times after chemotherapy exposure. C. Average number of nuclear fragments observed before and at different times after the treatment. Error bars represent ± one standard deviation.

Figure 4. Average values of quantitative ultrasound spectral parameters obtained for AML-5 cell samples in vitro using ~7 MHz ultrasound

Changes in the A. MBF, B. spectral slope and C. 0-MHz intercept parameters are shown at different times after chemotherapy exposure. Trends in the D. MBF, E. spectral slope and F. 0-MHz intercept parameters are demonstrated as functions of proportions of apoptotic cells mixed with untreated cells. Error bars represent ± one standard error.

Figure 5. In vivo study on AML-5 tumour xenografts

A–E: Representative data acquired from control and treated tumours. A. ultrasound B-mode images with ROI parametric overlays of the MBF parameter. The scale bar represents ~1 mm, and the color bar represents a scale encompassing ~40 dBr. B, C. light microscopy images of H&E stained slides at low and high magnifications. D, E. light microscopy images of TUNEL stained slides at low and high magnifications. The scale bar represents ~1 mm and ~20 μm in the low and high magnification images, respectively. F. Average values measured for the sizes of the cells and their nuclei before and after the treatment. G, H. Average values of the MBF and 0-MHz intercept parameters obtained before and after the treatment using ~7 MHz ultrasound. Error bars represent ± one standard error.