PET imaging of apoptosis and necrosis
Cell death can be either uncontrolled (necrosis, oncosis) or programmed (apoptosis). Both types of cell death are often present after trauma and in hypoxic tissue, for example in tumours and in severe ischemia (De Saint-Hubert et al., 2009; Reshef et al., 2010). Many imaging methods can detect both types, which is advantageous for example in cancer treatment studies, but separating viable and non-viable tissue is useful for example after severe ischemia.
Apoptosis may be induced by intrinsic (mitochondrial) or extrinsic (receptor-mediated) pathways. Both pathways lead to the activation of a cascade of cysteine aspartic acid proteases (caspases) which break down cell proteins in a controlled fashion. Cellular components are packaged for phagocytosis, in order to prevent any inflammatory reaction. Cytoplasmic [Ca2+] is an important signalling factor in apoptosis, as Ca2+ is normally stored in endoplasmic reticulum, lysosomes, and mitochondria, but can be released from there via certain ion channels or due to cellular damage. Apoptosis requires ATP, and if ATP is not available, cell turns to necrosis. In necrosis, cell membrane loses its integrity, causing cells to swell and burst, releasing cell contents, including proteolytic enzymes and aminophospholipids.
Phosphatidylserine (PS) is a phospholipid, and component of the cell membrane. In healthy cells, ATP-dependent aminophospholipid translocase (flippase) keeps PS on the cytosolic side of the membrane. In apoptotic cells flippase is less functional, but instead scramblase enhances the flipping of PS between the both sides of cell membrane. Macrophages identify the PS as a signal to start phagocytosis, but simultaneously the secretion of immunosuppressants. Blood platelets can trigger the clot formation by exposing PS on their outer surface.
Phosphatidylethanolamines (PE) are an abundant class of phospholipids in the cell membranes. Like PS, in healthy cells, PE resides predominantly in the inner leaflet of the cell membrane, but is externalized in dying cells. Normally, the external side contains mostly phosphatidylcholine and sphingomyelin.
Overall cell death imaging
PS exposition is evident in all types of cell death, and common in tumour cells, infection and inflammation, thrombosis, and during blood clot formation. Many pathogens expose PS on their surface to bind and enter the host cells (apoptotic mimicry); therefore PS imaging is not specific for cell death, but it may still be useful when these limitations are taken into consideration.
Also the exposure of PE on the cell surface has become an attractive target for the molecular imaging of cell death using SPECT and PET (Elvas et al., 2017). Duramycin binds PE, but currently developed 18F-labelled duramycin tracers have not been promising for in vivo PET imaging.
Annexin A5 (Annexin V, AnxV) is a protein with high affinity to PS. It is usable in in vitro and ex vivo studies, and has been labelled with 68Ga, 18F, 11C, and 64Cu (Lehner et al., 2012; Hu et al., 2012; Cheng et al., 2013; Grafström and Stone-Elander, 2014; Wuest et al., 2015; Perreault et al., 2016). Due to its relatively large size, Ca2+-dependence, and non-optimal kinetics, smaller molecules mimicking Annexin A5 are being developed for in vivo imaging.
Annexin B1 (AnxB1), another member of Annexins family, has high affinity toward the head group of PS. 18F-labeled AnxB1 has shown promise for apoptosis imaging (Wang M-W et al., 2013).
Synthetic bis(zinc(II)-dipicolylamine) (DPAZn2) coordination complexes have selective affinity for anionic membranes (Rice et al., 2016), such as PS enriched membranes. Several DPAZn2-based probes have been used for optical imaging, and tracers for SPECT and PET imaging have been developed (Wang H et al., 2013; Sun et al., 2015; Li et al., 2015).
Caspases are intracellular enzymes that are activated during apoptosis, and therefore provide a specific target for apoptosis imaging. Caspase tracers need to be able to penetrate the cell membrane to be useful, and may therefore be affected by changed perfusion and transport (Machulla, 2015).
The caspase substrate based PET tracer [18F]CP18 is capable of detecting the activity of caspase-3/7, two key executioner proteases in the apoptosis pathway, through selective cleavage of the ligand by the activated proteases and its subsequent accumulation in apoptotic cells. In vitro and in vivo models suggest that [18F]CP18 uptake is specifically related to apoptosis but not necrosis (Su et al., 2013; Rapic et al., 2017). [18F]C-SNAT is another caspase-3/7 substrate; after cleavage, it undergoes intracellular cyclization reaction leading to trapping inside the cells (Palner et al., 2015; Witney et al., 2015).
Isatin sulphonamides are specific caspase-3 and caspase-7 inhibitors. Several positron emitting isotope labelled analogues have been developed (Limpachayaporn et al., 2013; Médoc et al., 2016), including [18F]ICMT-11 (Nguyen et al., 2009; Challapalli et al., 2013), [18F]WC-II-89 (Zhou et al., 2006), [18F]WC-IV-3 (Chen et al., 2009), [18F]WC-98 (Chen et al., 2009), [18F]WC-4-116 (Thukkani et al., 2016), and [18F]CbR (Faust et al., 2007).
Caspase-3 activity can be monitored using cTK reporter in in vivo tumour models (Wang F et al., 2014; Wang Z et al., 2015); active caspase-3 splices the cyclic reporter into active thymidine kinase, and its activity can then be measured using its substrate [18F]FHBG.
[18F]ML-10, 2-(5-18F-pentyl)-2-methyl-malonic acid, which can cross depolarized cell membranes, but cannot enter the cytoplasm of healthy cells (Höglund et al., 2011; Oborski et al., 2014; Demirci et al., 2017). [18F]ML-10 is released from ruptured cells.
Mitochondrial membrane potential
The collapse of mitochondrial membrane potential plays a key role in apoptosis. Voltage-sensitive radiotracers, such as [18F]FBnTP, have potential to be used in apoptosis imaging, although tracer efflux by multidrug-resistance proteins may limit their usability (Reshef et al., 2010).
PD-1 (programmed cell death 1) is a member of B7-CD28 family of proteins that can be expressed on the surface of activated T-cells, B-cells, dendritic cells, and macrophages. Antigen presenting cells and many tumour cells express on their surface its natural ligands, PD-L1 and PD-L2, that inhibit T-cell activation and effector function. Immunotherapies have been developed to block the PD-1/PD-L1 system to enhance cytotoxic T-cell and NK cell response. 89Zr-labelled nivolumab could be useful in imaging activated T-cells in tumours and in autoimmune diseases (England et al., 2018).
Several SPECT tracers for detecting necrosis have been developed. Necrosis can be detected by targeting molecules that are confined into intracellular space in healthy and apoptotic cells, but that are exposed when cells burst uncontrollably.
Pamoic acid is an inhibitor of DNA polymerase β. [68Ga]bis-DOTA-PA is a potential tracer for specific necrosis imaging (Prinsen et al., 2013).
- PET imaging of hypoxia
- PET imaging of infection and inflammation
- Synaptic density
Bauwens M. In vivo apoptosis imaging using site-specifically 68Ga-labeled Annexin V. Methods Mol Biol. 2016; 1419: 17-26.
De Saint-Hubert M, Prinsen K, Mortelmans L, Verbruggen A, Mottaghy FM. Molecular imaging of cell death. Methods 2009; 48: 178-187.
Elvas F, Stroobants S, Wyffels L. Phosphatidylethanolamine targeting for cell death imaging in early treatment response evaluation and disease diagnosis. Apoptosis 2017; 22(8): 971-987.
Höglund J, Shirvan A, Antoni G, Gustavsson S-Å, Långström B, Ringheim A, Sörensen J, Ben-Ami M, Ziv I. 18F-ML-10, a PET tracer for apoptosis: first human study. J Nucl Med. 2011; 52(5): 720-725.
Machulla H-J. Imaging of apoptosis: the need to distinguish tracer uptake rate from regional contribution of blood flow. J Nucl Med. 2015; 56(9): 1300-1301.
Mor G, Alvero AB (eds.): Apoptosis and Cancer, 2nd ed. Humana Press, 2015. doi: 10.1007/978-1-4939-1661-0.
Puthalakath H, Hawkins CJ (eds.): Programmed Cell Death. Humana Press, 2016. doi: 10.1007/978-1-4939-3581-9.
Rapic S, Vangestel C, Elvas F, Verhaeghe J, den Wyngaert TV, Wyffels L, Pauwels P, Staelens S, Stroobants S. Evaluation of [18F]CP18 as a substrate-based apoptosis imaging agent for the assessment of early treatment response in oncology. Mol Imaging Biol. 2017; 19(4): 560-569.
Reed JC, Green DR (eds.): Apoptosis - Physiology and Pathology. Cambridge University Press, 2011. ISBN 978-0-521-88656-7.
Reshef A, Shirvan A, Akselrod-Ballin A, Wall A, Ziv I. Small-molecule biomarkers for clinical PET imaging of apoptosis. J Nucl Med. 2010; 51: 837-840.
Rice DR, Clear KJ, Smith BD. Imaging and therapeutic applications of zinc(ii)-dipicolylamine molecular probes for anionic biomembranes. Chem Commun. 2016; 52(57): 8787-8801.
Su H, Chen G, Gangadharmath U, Gomez LF, Liang Q, Mu F, Mocharla VP, Szardenings AK, Walsh JC, Xia CF, Yu C, Kolb HC. Evaluation of [18F]-CP18 as a PET imaging tracer for apoptosis. Mol Imaging Biol. 2013; 15(6): 739-747.
Sun T, Tang G, Tian H, Hu K, Yao S, Su Y, Wang C. Positron emission tomography imaging of cardiomyocyte apoptosis with a novel molecule probe [18F]FP-DPAZn2. Oncotarget 2015; 6(31): 30579-30591.
Wang F, Wang Z, Hida N, Kiesewetter DO, Ma Y, Yang K, Rong P, Liang J, Tian J, Niu G, Chen X. A cyclic HSV1-TK reporter for real-time PET imaging of apoptosis. Proc Natl Acad Sci USA 2014; 111(14): 5165-5170.
Wang H, Tang X, Tang G, Huang T, Liang X, Hu K, Deng H, Yi C, Shi X, Wu K. Noninvasive positron emission tomography imaging of cell death using a novel small-molecule probe, 18F labeled bis(zinc(II)-dipicolylamine) complex. Apoptosis 2013; 18(8): 1017-1027.
Wang M-W, Wang F, Zheng YJ, Zhang YJ, Zhang YP, Zhao Q, Shen CK, Wang Y, Sun SH. An in vivo molecular imaging probe 18F-Annexin B1 for apoptosis detection by PET/CT: preparation and preliminary evaluation. Apoptosis 2013; 18(2): 238-247.
Wang X, Feng H, Zhao S, Xu J, Wu X, Cui J, Zhang Y, Qin Y, Liu Z, Gao T, Gao Y, Zeng W. SPECT and PET radiopharmaceuticals for molecular imaging of apoptosis: from bench to clinic. Oncotarget 2017; 8(12): 20476-20495.
Zeng W, Wang X, Xu P, Liu G, Eden HS, Chen X. Molecular imaging of apoptosis: from micro to macro. Theranostics 2015; 5(6): 559-582.
Created at: 2017-09-13
Updated at: 2018-07-25
Written by: Vesa Oikonen