In vivo magnetic resonance imaging of treatment-induced apoptosis

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.

Figure 1

Flow cytometry analyses indicate the occurrence of apoptosis in cetuximab-treated DiFi cell pellets and a corresponding decrease in cell size. (A) Histograms of the emission light intensity of PI labelled cells (PI-A). (B) Box-and-whisker plots of the FSC-W for DiFi and HCT116 cell pellets treated with 20 nM cetuximab for 0, 24, and 48 hrs. The sample size of each cohort is 10000. For all the Box-and-whisker plots, the 25^th–75^th percentiles are blocked by the box, the black and red bands inside the box are the median and mean, respectively, and the whiskers mark the SD. ****P < 0.0001 as measured by one-way ANOVA with a FDR (False Discovery Rate) posttest.

Figure 2

TDSI-derived restriction size detects decreases in cell size for cetuximab-treated DiFi cells. (A,B) Fitted d (A), D_f→inf (B), D_f→0 (C), and PGSE-derived ADC (D) for DiFi and HCT116 cell pellets treated with 20 nM cetuximab for 0, 24, and 48 hours. *P < 0.05 as measured by one-way ANOVA with a FDR (False Discovery Rate) post test.

Figure 3

PGSE-derived ADC map, and TDSI-derived parametric maps (restriction size d, D_f→inf, and D_f→0) of a representative slice through a non-treated DiFi tumor, overlaid on T2-weighted MR images.

Figure 4

(A–E) Percentage changes in tumor volume (A), ADC obtained from PGSE (B), and three fitted spectral parameters (d, D_f→inf, and D_f→0) for cetuximab/PBS-treated DiFi and cetuximab-treated HCT116. (F) Volume fractions of late-stage apoptotic regions defined by model selection between the constant ADC model and tumor ADC model described in Eq. 3. *P < 0.05 as measured by one-way ANOVA with a FDR (False Discovery Rate) posttest.

Figure 5

(A) Caspase-3 stained histological images of DiFi tumor tissues treated with PBS and Cetuximab at day-0 (no treatment), day-4 (2 treatments), and day-8 (4 treatments), x2. (B) Na+/K+-ATPase stained histological images for tissue regions consisting either of active tumor cells, early stage apoptotic cells, or late stage apoptotic cells. Nuclei were visualized with DAPI (blue). Note that all the cells express high level, relative evenly distributed Na+/K+-ATPase (green) on their plasma membranes.