Development of a novel imaging platform for the detection of apoptotic cells

Abstract

In the human body, billions of cells die every day, a cell death that is termed apoptosis. Clearance of these cells is crucial for the maintenance of homeostasis and dysregulation leads to inflammation and cancer. The extent of apoptosis within tissues is indicative of development of disease and correlates with the efficacy of therapies that reverse its progression. However, current optical methods for the detection of apoptosis are incompatible with in vivo physiological conditions. Molecules that target intracellular changes (e.g. caspase activation, DNA fragmentation) have slow binding kinetics and depend on the permeabilization and fixation of cells. In contrast, reagents targeting the extracellular alterations that accompany apoptosis exhibit improved kinetics and have the potential for live-cell imaging. However, most of those reagents do not permit imaging in areas with low free calcium ion concentrations that is common in diseased tissues.

In previous literature, amphipathic peptides have been exploited as a monitor for alterations in the membrane phospholipid composition. The interactions of the peptides with a membrane is dependent on their sequence, charge, hydrophobicity, amphiphilicity as well as secondary structure. The phospholipid bilayer is mainly composed of phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PtdSer) and sphingomyelin. During the apoptotic process the lipid distribution changes from PC and sphingomyelin to PtdSer and PE as the main components of the extracellular leaflet. Thus, this thesis aims to exploit the interaction between amphipathic peptides with distinct lipid motifs exposed on the surface of apoptotic cells. Therefore, we developed a small library of fluorogenic peptides with amino acid sequences varying the amount of hydrophobic and hydrophilic residues. In order to confer suitable stability and biodistribution of the peptides in vivo, the peptides were kept to seven amino acids and cyclised. Screening of the library provided insights into the motifs needed in the peptide to specifically stain apoptotic/necrotic cells. Experiments demonstrated the necessity of three positively-charged amino acid residues combined with three hydrophobic residues to acquire and maintain stable binding to apoptotic cells. The lead peptide with these characteristics was called “Apo-15”. Using flow cytometry to analyse the kinetics and reversibility of peptide binding to apoptotic cells in real-time, I demonstrated the necessity of three positively charged amino acid residues combined with three hydrophobic residues to confer specific and stable binding. Moreover, experiments confirmed interactions of the peptide with the apoptotic cells to be dependent on the exposure of PtdSer on the extracellular surface. Using ELISA-like plate-based assays with lipid monolayers the preferential binding of the lead peptide to PtdSer was shown.

Further competition of PtdSer-ligand Annexin V with the lead peptide and the lack of its binding to a cell line lacking the exposure of PtdSer using flow cytometry confirmed PtdSer as a target. The lead peptide chosen for further experiments excelled over other apoptotic detection reagents in its ion-independence as well as binding kinetics and compatibility with real-time imaging. Experiments also demonstrated the use of this probe to detect apoptosis using multiple fluorescence-based imaging modalities, both in vitro and in mouse models of lung inflammation and cancer in vivo. Ex vivo studies of the in vivo administered lead peptide showed its use for staining apoptotic cells in bronchoalveolar lavage as well as lung and tumour tissue. Furthermore, based on the lead peptide we designed a red-shifted version of the probe for comparison in real-time imaging, termed “ApoRed”. Using flow cytometry and microscopy, the red-shifted derivative was compared to the lead peptide. Experiments have shown the compatibility of Apo-15 as well as ApoRed with higher signal-background in ApoRed.

Both probes were successfully applied to intravital imaging for in vivo in real-time detection of apoptosis using two photon and spinning disk microscopy. Together the work in this thesis has highlighted the utility of small amphipathic peptides as reagents for imaging the membrane alterations associated with apoptosis and have demonstrated their potential use in monitoring the effectiveness of drug treatments in inflammatory diseases and cancer.