Abstract 5757: Magnetic Resonance Imaging with Iron-Oxide Labeling Detects Differential Cell Survival after Doxorubicin Exposure in Cardiac Myocytes, Fibroblasts, and Stem Cells

Abstract

Heart failure caused by anthracycline chemotherapy, e.g. doxorubicin (DOX), is preceded by significant cell apoptosis. Early, non-invasive detection of cardiac cell death may improve patient outcomes by enabling real-time adjustment of chemotherapy. To this end, Annexin V protein (ANX) binds to membrane-bound phosphatidylserine, which is externalized in early apoptosis. We tested whether ANX conjugated to superparamagnetic iron oxide (SPIO) could detect and quantify apoptosis via T2-weighted magnetic resonance imaging (MRI). ANX was purified and conjugated to SPIO. Cultured, neonatal rat cardiac myocytes (CMs) and fibroblasts (FBs), and mouse mesenchymal stem cells (mMSCs) were exposed to DOX (1uM) for varying times, then stained with ANX-SPIO, or with fluorescent ANX (ANX-FLUOS, Roche) or TUNEL as controls. In vitro MRI (3 Tesla, GE Excite, WI) was performed on plated cells using a gradient echo (GRE) sequence (see Table ). MRI signal after ANX-SPIO incubation correlated highly with the number of ANX-FLUOS or TUNEL positive cells after exposure to varying durations of DOX (R=0.91 ANX-FLUOS vs. ANX-SPIO, n=5; R=0.80 TUNEL vs. ANX-SPIO, n=4; p<0.05). ANX-SPIO signal was detectable by MRI from as few as 15–20 apoptotic mMSCs. CMs exhibited the highest sensitivity to DOX (see Table ), with peak apoptosis after 30 minutes of DOX exposure. Conversely, cardiac FBs were resistant to DOX for 22 hours, and mMSCs showed intermediate susceptibility to DOX. Competition binding between ANX-SPIO and ANX-FLUOS confirmed the specificity of ANX-SPIO for apoptotic cells in culture. In vitro MRI of a magnetically-labeled marker of apoptosis (ANX-SPIO) can accurately detect cell death in culture with high sensitivity. MRI of ANX-SPIO can also resolve differential susceptibility to DOX in CMs, FBs, and mMSCs. This compound may hold promise for in vivo MRI detection of early anthracycline-induced cell death in the myocardium. This research has received full or partial funding support from the American Heart Association, AHA Western States Affiliate (California, Nevada & Utah). Cell-Type Specific Susceptibility to Doxorubicin Assessed by MRI