Fluorescence Lifetime Imaging of Apoptosis

Abstract

Genetically-encoded fluorescence resonance energy transfer (FRET) reporters are powerful tools to analyze cell signaling and function at single cell resolution in standard two-dimensional cell cultures, but these reporters rarely have been applied to three-dimensional environments. FRET interactions between donor and acceptor molecules typically are determined by changes in relative fluorescence intensities, but wavelength-dependent differences in absorption of light complicate this analysis method in three-dimensional settings. Here 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 two-dimensional 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 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.

A Bifunctional MAPK/PI3K Antagonist for Inhibition of Tumor Growth and Metastasis

“…A2058 melanoma cells were infected with lentivirus pLVX-EF1aLuc2-IRES-blast previously described (14) and selected for blastacidin resistance by supplementing the media with 10 μg/ml blastacidin (Gibco).…”

Apoptosis detection via automated algorithms to analyze biomarker translocation in reporter cells

“…In both cases, a method to identify the effectiveness of a drug is vital, and the ability to streamline the screening into a robust, reliable, high-throughput process is necessary. There are several assays available commercially and/or for research purposes from simple live-dead assays and surface apoptosis markers such as Annexin-V, to cytosolic monitoring of apoptosis through caspase reporter biomarkers (Carpenter et al, 2006;Gelles & Chipuk, 2016;Helmy & Azim, 2012;Lamprecht, Sabatini, & Carpenter, 2007;Saraste & Pulkki, 2000;Saraste, 1999;Xiao, Gibbons, Luker, & Luker, 2015;Ziraldo, Link, Abrams, & Ma, 2014. Most apoptosis assays are end-point assays lacking abilities to report dynamic drug responses temporally and intracellular spatially.…”

In vitro and in vivo phasor analysis of stoichiometry and pharmacokinetics using near-infrared dyes

“…Alternative methods such as rapid lifetime determination (RLD 27 ) and minimal fraction of interacting donor 28 have been developed to circumvent these limitations, but these methods have not been tested with short-lifetime NIR dyes in the presence of broad impulse response function (IRF) such as encountered in in vivo measurements 29 . In these situations, an approach such as phasor analysis that does not rely on specific decay models and does not focus on extracting lifetimes, could be particularly useful due to its speed and ease of use as demonstrated next [30][31][32][33][34][35][36][37][38][39][40] .…”

“…Finally, precise quantification of the respective fractions of two or more components can be performed, provided the location of known species' phasor location can be determined. This method has been successfully applied in a number of in vitro studies using fluorophores emitting in the visible range of the spectrum and characterized by fluorescence lifetimes that are long compared to the width of the instrument response function (IRF) [35][36][37][38][39][40] Importantly, these studies have so far been exclusively performed in vitro where very little scattering takes place.…”