October 15, 2017 (Vol. 37, No. 18)
Andrew L. Niles Senior Research Scientist Promega
Kevin R. Kupcho Research Scientist Promega
Continuous Assessment of Cell Death Mechanism of Action Using Annexin V
Cell death in vitro can be induced by several distinct mechanisms depending upon the type and potency of the stimulus. Biological variables present a challenge to rapid and efficient characterization of the mode of cell death for a biologic or a new chemical entity during the process of drug discovery.
Although assay methods to study cell death pathways have improved in recent years, with respect to sensitivity, miniaturization, and ease-of-use, kinetics of cell death have been largely ignored in favor of convenience as provided by often arbitrary endpoint measures. Endpoint measurement approaches miss important information relating to the cytotoxic phenotype.
Conversely, real-time (non-endpoint), live-cell assay methods are better suited for characterizing the kinetics and magnitude of a cytotoxic response. Although elegant methods have been devised using electrical impedance and optical imaging, they are limited in throughput and require both specialized instrumentation and training, and costly consumables.
We have developed a real-time, plate-based method that is accessible to any laboratory with a multimode (i.e., fluorescence and luminescence) reader. The new method is called RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay.
Established Biomarker, New Approach
Viable cells ensure their physical and functional integrity through a highly ordered membrane structure consisting of an array of proteins and phospholipids. One such class of proteins is phosphatidylserine (PS), which is actively maintained in an asymmetric orientation at the cytosolic membrane surface of healthy cells. During apoptosis induction, PS translocates to the cell surface. In vitro, externalized PS represents a reliable and quantifiable biomarker of apoptosis.
Fluorescently labeled annexin V is a traditional imaging agent for detecting PS translocation because of its highly specific and robust calcium ion-dependent affinity for PS. This technique is limiting, though, as it is restricted to endpoint measurements and requires removal of unbound annexin V probe by repeated washes of cell samples prior to analysis by microscopy or flow cytometry.
For our improved method, we created annexin V-fusion proteins, which act as a complementation reporter, and that contains two different subunits of an improved small and highly sensitive NanoBiT® luciferase enzyme. In the absence of externalized PS, the annexin V-fusion proteins have only modest affinity for each other, and luminescence from random complementation of the luciferase enzyme is practically nonexistent.
The assay is homogeneous with no requirement for washes. When PS exposure occurs during apoptosis, the Annexin V-NanoBiT protein pairs are drawn to close proximity for complementation after binding to PS rafts. Complementation reconstitutes a functional luciferase protein reporter molecule. Luminescence, as a result of this pairing, generates a magnitude of response proportional to PS exposure.
Figure 1. How the assay works. RealTime-Glo™ Annexin V Apoptosis Assay uses Annexin V fusion proteins containing two complementary subunits (Annexin V-LgBiT and Annexin V-SmBiT) of NanoBiT® luciferase. When in close proximity, the LgBiT and SmBiT subunits come together to form a functional NanoBiT® luciferase. Left: Healthy Cell. Detection reagent contains Annexin V-LgBiT and Annexin V-SmBiT (NanoBiT) fusion proteins and a profluorescent DNA dye. Middle: Early Apoptosis. Luminescence remains low until PS exposure brings the annexin fusion proteins close together, forming a functional NanoBiT® luciferase. Right: Secondary Necrosis. A fluorescent signal is generated upon loss of membrane integrity during late-stage apoptosis, when the DNA dye can enter the cell.
In cases of secondary necrosis (the eventual fate of apoptosis with in vitro systems) or in cases of other forms of necrosis that cause gross loss of membrane integrity, PS-driven Annexin V-NanoBiT complementation can occur either on the external or internal membrane surface. Figure 1 illustrates three stages of cells in the apoptotic process: healthy, early apoptosis, and secondary necrosis.
In the new homogeneous method, a 2X reagent is added in the cell-culture medium of choice using the Annexin V-NanoBiT protein fusions, a luciferase substrate, and an optional pro-fluorescent cytotoxicity probe. Additional calcium chloride is provided to facilitate a maximized PS-annexin binding interaction. Reagent can be added directly to cell cultures at the same time as test compound is added.
Figure 2 demonstrates an apoptotic response, initiated by an on-market antibody-drug conjugate, trastuzumab emtansine, ultimately resulting in secondary necrosis. Figure 3 illustrates a 48-hour kinetic trace of PS exposure and necrosis signal following treatment with the same drug.
Real-Time Biomarker Detection during Extended Exposures
Our improved, real-time annexin-based method is technically possible due to the novel chemical attributes of the luminogenic substrate and fluorogenic cytotoxicity probe. An esterified form of the luciferase substrate is delivered in great molar excess as part of the assay reagent, and becomes an active form of the substrate as it is de-protected. Esterase activity is inherent in viable cells as well as some serum supplements, and substrate de-protection progresses at a steady and sustained rate in viable cells. In most instances, the active substrate is made available even during incubations up to 48 hours in length.
An optional, unique fluorogenic cytotoxicity probe with affinity for DNA serves as a measure for loss of membrane integrity due to necrosis. The viable cell-impermeant probe produces nominal fluorescence signal in the absence of necrotic cells, but when cells lose membrane integrity, it complexes with exposed DNA, resulting in a substantial increase in fluorescence. This real-time necrosis probe in the reagent allows sequential measure of luminescence (as a result of PS exposure) and of fluorescence (as a result of necrosis) in the same sample to monitor cytotoxic progression.
Use Time to Your Advantage
Monitoring real-time PS exposure and necrosis during the induction of cell death may provide important contextual clues relating to mechanisms of action. For instance, strong luminescence signals resulting from PS exposure without measurable increases in fluorescence indicates classical early apoptotic phenotype. Likewise, strong luminescence signals with modest fluorescence increases may indicate a later maturing, dose-dependent apoptotic response. Conversely, strong and concurrent increases in both luminescence and fluorescence immediately following a stimulus would be consistent with primary necrosis. Therefore, relative kinetic emergence of these two biomarkers can help profile putative actions of a compound or biologic.
One can multiplex RealTime-Glo Annexin V Apoptosis and Necrosis Detection Assay with several value-added endpoint chemistries to gather additional information regarding mechanisms of cell death. For instance, multiplexing with cell viability reagents (e.g., Cell-Titer-Glo® or CellTiter-Fluor™) can either provide inversely correlated measures of cytotoxicity (by loss of signal) or reveal hidden cytostatic effects mediated by test agents. Similarly, caspase activity reagents (e.g., Caspase-Glo®) can provide orthogonal proof of the apoptotic phenotype when counter-confirmation is required. Collection of these multiplexed signals is possible due to either spectral compatibility of the individual signals or quenching attributes associated with reagent formulations.
The real-time, bioluminescent assay method for apoptosis and necrosis described here provides full functionality of conventional (endpoint) annexin methods while requiring substantially less effort and being plate-based friendly. Time-resolved nature of the assay allows for dose-dependent characterization of cytotoxic effects while providing kinetic resolution of cell death mechanisms of action. Finally, multiplexing the assay with other endpoint cell health assays can provide additional complementary or orthogonal data.