High-frequency ultrasound detection of cell death: Spectral differentiation of different forms of cell death in vitro.

Maurice M. Pasternak,Gregory J. Czarnota,Ali Sadeghi-Naini,Michael L. Oelze,Shawn M. Ranieri,Anoja Giles,Michael C. Kolios

doi:10.18632/oncoscience.319

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.

Highlights

Cell death introduces significant alterations in physical and morphological characteristics of cells and nuclei [1]
Quantitative analysis of the RF signal was found to be effective in differentiating forms of cell death at experimental times and results were co-incident with observed morphological alterations from histology
Quantitative ultrasound spectral and backscatter parameters including the spectral slope, the mid-band fit (MBF), the 0-MHz intercept, the average acoustic concentration (AAC), and the average scatterer diameter (ASD) have been previously used to characterize biological tissues [26, 34, 35, 36, 37, 38]. Most of these studies have focused on one or two forms of cell death, most commonly apoptosis, which is not the only form of cancer cell death induced by anti-tumor treatment. This investigation expanded the use of High frequency ultrasound (HFUS) to compare and characterize other modes, including oncosis, mitotic arrest, and heat death in order to demonstrate its capacity for differentiating modes of cell death

Summary

Introduction

Cell death introduces significant alterations in physical and morphological characteristics of cells and nuclei [1] It can be categorized into different forms including apoptosis (programmed cell death), oncosis, mitotic arrest (death in mitosis), and coagulative cell death (e.g., due to heating). Even at a molecular level, recent studies have indicated that markers such as TUNEL positivity [2, 3], or the Annexin V positive / propidium iodide negative phenotypes [4, 5] are not unique to apoptosis These various forms of cell death have distinct morphological features which are associated with potentially-unique viscoelastic properties www.impactjournals.com/oncoscience that alter their acoustic properties and ultrasound scattering. Necrosis should not be considered a form of cell death, but rather as the end stage to any cell death process, as outlined by Majno and Joris [1]

Methods

Results

Conclusion