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.

Computer aided prognosis for cell death categorization and prediction in vivo using quantitative ultrasound and machine learning techniques

“…The proposed CAP system has the potential to be adapted to clinical applications 85,86 for noninvasive therapeutic cancer response assessment, a step toward personalized medicine. To this end, in addition to the requirement of using QUS methods at conventional frequencies, 10,18,58 some changes will be required to extend the proposed methods to patient data. These changes will include: performing image acquisition several times during the course of treatment, e.g., on weeks 1, 4, and 8, for longitudinal monitoring of the therapy effects; a different regime for histological analysis, as it only provides final treatment response, not the level of cell death during the course of treatment; and likely the requirement to employ techniques for learning from imbalanced data, as the number of responders is usually much higher than the number of nonresponders in clinical studies.…”

“…The applicability of QUS methods to detect different modalities of cell death, including mitotic arrest and apoptosis, has been demonstrated in several in vitro, in situ, and in vivo studies with multiple tumor types and using different therapeutic modalities. 4,6,8,9,12,[15][16][17][18] The sensitivity of highfrequency-ultrasound backscatter intensity to cell death resulting from cancer treatment was initially demonstrated by Czarnota et al in vitro 8 and then in vivo. 12 The research was extensively followed by in vitro and in vivo studies demonstrating the capability of high-frequency QUS methods in detecting and monitoring cell death occurring as a result of cancer treatment.…”

“…These studies used restricted settings, such as the same training and test sets to demonstrate the proof of principle of the proposed QUS methods. 9,10,17,18,20,57,58 However, state-of-the-art texture methods, advanced machine learning techniques, and supervised learning in a realistic setting (such as leave-one-subject-out validation scheme), as presented in this study, are needed in order to design a complete computer-aided-prognosis system for the early prediction (within hours after the start of treatment) of cell death levels reliably, and automatically, both at discrete and continuous levels. As a summary, the main contributions of this study are as follows:…”

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

“…The use of such cells was selected as a crude approximation of cell-dense tumors with minimal vasculature. We have previously investigated the apoptotic death response of this cell line using quantitative ultrasound validated by histology [61]. …”

“…Centrifuged cell samples were scanned in stainless steel wells [26, 61]. Three samples were scanned for each treatment condition, along with an untreated control sample.…”