Abstract
Imaging apoptosis could provide an early and specific means to monitor tumor responses to treatment. To date, despite numerous attempts to develop molecular imaging approaches, there is still no widely-accepted and reliable method for in vivo imaging of apoptosis. We hypothesized that the distinct cellular morphologic changes associated with treatment-induced apoptosis, such as cell shrinkage, cytoplasm condensation, and DNA fragmentation, can be detected by temporal diffusion spectroscopy imaging (TDSI). Cetuximab-induced apoptosis was assessed in vitro and in vivo with cetuximab-sensitive (DiFi) and insensitive (HCT-116) human colorectal cancer cell lines by TDSI. TDSI findings were complemented by flow cytometry and immunohistochemistry. Cell cycle analysis and flow cytometry detected apoptotic cell shrinkage in cetuximab-treated DiFi cells, and significant apoptosis was confirmed by histology. TDSI-derived parameters quantified key morphological changes including cell size decreases during apoptosis in responsive tumors that occurred earlier than gross tumor volume regression. TDSI provides a unique measurement of apoptosis by identifying cellular characteristics, particularly cell shrinkage. The method will assist in understanding the underlying biology of solid tumors and predict tumor response to therapies. TDSI is free of any exogenous agent or radiation, and hence is very suitable to be incorporated into clinical applications.
Highlights
Apoptosis, or a programmed cell death, is the primary mechanism by which unwanted cells are selectively eliminated during growth, differentiation, and maintenance of tissue homeostasis
Conventional Diffusion-weighted MRI (DWI) quantifies diffusion in terms of an apparent diffusion coefficient (ADC), which has been found widely useful for monitoring anti-cancer therapies[24,25] but lacks specificity as it is influenced by several factors including the tortuosity of extracellular spaces, tissue cellularity, and the integrity of cellular membranes[22]
The emission light intensity of propidium iodide labelled cells (PI-A) is proportional to the amount of DNA present in the cells, so the percentage of cells in different phases of the cell cycle can be calculated from the histogram of PI-A39
Summary
A programmed cell death, is the primary mechanism by which unwanted cells are selectively eliminated during growth, differentiation, and maintenance of tissue homeostasis. Optical imaging has been shown to provide real-time imaging of therapy-induced apoptosis[13,14], but suffers poor penetration into biological tissues, limiting its applicability to superficial tumors. High-frequency ultrasound imaging may characterize apoptosis in real-time by detecting sub-cellular nuclear changes[15,16] but it cannot be used in many tumor sites due to its low focal detection range in tissues. Apoptosis is a highly regulated and controlled process with characteristic cellular morphologic changes, such as cell membrane blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and DNA fragmentation. These morphologic changes are distinct from those that arise in necrosis, and have been considered as hallmarks of apoptosis[23].
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