Molecular magnetic resonance imaging: implications to the detection of apoptosis

Introduction/Theory: Molecular imaging is emerging as a potential tool for monitoring treatment response in the management of cancer patients. Recent cell biology as well as oncology research has focused on investigating apoptosis or programmed cell death as a means of measuring these induced effects and detecting response. A hallmark of early stage apoptosis is the externalization of phosphatidylserine (PS). The human protein annexin V with its high binding affinity for PS has been used ubiquitously in the biochemistry apoptosis FITC assay. As previously demonstrated in cardiomyocyte cells, annexin V + SPIO nanoparticles can provide sufficient MRI T2 and T2* contrast enhancement for in-vivo detection of apoptosis. The aim of the present study was to demonstrate the feasibility of imaging the therapeutic response of cancer cells by establishing a quantitative relationship between T2 enhancement and apoptotic extent. Specifically, heat shock induced apoptosis in human leukemia HL60 cells in-vitro was probed by tagging with Annexin V+SPIO and subsequent analysis of the transverse relaxation (T2) measured with a multi echo spin echo MRI acquisition. The results determined a linear relationship between spin-spin relaxivity (R2) and the percentage apoptosis in the cell population. Methods: In order to detect early stage apoptosis, a number of methods were used. 100 percent apoptosis was induced in HL-60 (human leukemia) cells by placing cells in a 65oC water bath for 15 minutes. One hour, post treatment, the level of apoptosis was determined by staining with propidium iodide and annexin V-FITC. In order to generate cell populations consisting of varying percentages of apoptotic cells, cells treated at 65oC were mixed with untreated cells at known concentrations. The cells were analyzed by flow cytometry using a FACS Calibur flow cytometer (Becton Dickinson). The remainder of the cell population was stained with Annexin V MACS Microbeads (Mylteni Biotech) resuspended in a 50 μl volume of 1% carrageenan gel (Sigma Aldrich), placed in wells within a 384-well microplate, which was then analyzed by MRI (1.5 Tesla Seimens Avanto Clinical MRI scanner). A 16echo spin-echo imaging sequence was used with echo times (TE) of 15-300 ms. The regions of interest (ROI) used to extract T2 values consisted of approximately 68-70 pixels and R2 values were obtained by taking the inverse of an average of 5 ROIs taken from 5 separate wells. Standard deviations were recorded and accurately propagated to produce error in the relaxation times. The results from the apoptosis data obtained by flow cytometry were then correlated with the T2 relaxation rate from an MRI of the same population using SPIO molecules targeted to PS. A)