Abstract
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.
Highlights
Apoptosis, a common form of programmed cell death, is fundamental to cancer biology and therapy [1]
Caspase Reporter Shows Changes in Fluorescence Lifetime after Incubation with Proapoptotic Stimuli To develop a reporter for single-cell fluorescence lifetime imaging microscopy (FLIM) analysis of apoptosis in vitro and in vivo, we used a fluorescence resonance energy transfer (FRET) reporter based on the fusion of fluorescent proteins large Stokes shift (LSS)-mOrange and mKate2 with an intervening caspase-3 cleavage site [13]
We defined a region of interest (ROI) for the longer fluorescence lifetime of LSS-mOrange after caspase-3 cleavage of the reporter and loss of FRET with mKate 2 based on data from cells transfected with LSS-mOrange alone and without Bcl-2-associated X (Bax)
Summary
A common form of programmed cell death, is fundamental to cancer biology and therapy [1]. Resistance to apoptosis defines a hallmark feature of cancer initiation and progression, allowing cells to overcome cell-intrinsic and tissue-level safeguards against malignant transformation [2]. Apoptosis defines a common mechanism of cell death caused by most cancer chemotherapeutic drugs. In response to inciting events such as drug-mediated DNA damage, the blockade of pathways necessary for cell survival, or immunotherapy, cancer cells begin a well-characterized cascade of molecular events that involve the activation of caspases, a family of proteases [3]. Imaging caspase-3 activity provides a noninvasive, real-time method for quantifying apoptosis in response to environmental stresses and drugs in cell-based assays and living mice
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