Fluorescence Lifetime Imaging of Apoptosis.
Fluorescence Lifetime Imaging of Apoptosis.
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DOI：
10.18383/j.tom.2015.00163
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发表时间：
2015-12
期刊：
影响因子：
--
通讯作者：
Luker GD
中科院分区：
文献类型：
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作者：
Xiao A;Gibbons AE;Luker KE;Luker GD
Genetically encoded fluorescence resonance energy transfer (FRET) reporters are powerful tools for analyzing cell signaling and function at single-cell resolution in standard 2D cell cultures, but these reporters rarely have been applied to 3D environments. FRET interactions between donor and acceptor molecules typically are determined by changes in relative fluorescence intensities, but wavelength-dependent differences in light absorption complicate this analysis method in 3D settings. Herein we report fluorescence lifetime imaging microscopy (FLIM) with phasor analysis, a method that displays fluorescence lifetimes on a pixel-wise basis in real time to quantify apoptosis in breast cancer cells stably expressing a genetically encoded FRET reporter. This microscopic imaging technology allowed us to identify treatment-induced apoptosis in single breast cancer cells in environments ranging from 2D cell culture, spheroids with cancer and bone marrow stromal cells, and living mice with orthotopic human breast cancer xenografts. Using this imaging strategy, we showed that combined metabolic therapy targeting glycolysis and glutamine pathways significantly reduced overall breast cancer metabolism and induced apoptosis. We also determined that distinct subpopulations of bone marrow stromal cells control the resistance of breast cancer cells to chemotherapy, suggesting heterogeneity of treatment responses of malignant cells in different bone marrow niches. Overall, this study establishes FLIM with phasor analysis as an imaging tool for apoptosis in cell-based assays and living mice, enabling real-time, cellular-level assessment of treatment efficacy and heterogeneity.
