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 is activated in many neurodegenerative disease, including AD and PD.

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).

The C2A domain of synaptotagmin-1 binds PS with nanomolar affinity. In C2Am a cysteine residue has been added to enable site-specific labelling. ⁶⁸Ga and ¹⁸F labelled C2Am derivatives have been introduced for PET imaging (Bulat et al., 2020 and 2023).

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 radiopharmaceuticals for SPECT and PET imaging have been developed (Wang H et al., 2013; Sun et al., 2015; Li et al., 2015).

Imaging apoptosis

Caspase

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; Ostapchenko et al., 2019).

The caspase substrate based PET radiopharmaceutical [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; Xia 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); further development has provided [¹⁸F]C-SNAT4 with better stability (Chen et al., 2021). [⁶⁸Ga]Ga-TC3-OGDOTA is a bifunctional radiotracer that contains peptide helping passage through cell membranes; intracellular caspase-3 cleaves the peptide, leaving the label-carrying part of molecule trapped in the cells (Ostapchenko et al., 2019).

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.

Membrane depolarization

[¹⁸F]ML-10, 2-(5-¹⁸F-pentyl)-2-methyl-malonic acid, 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 necrotic, ruptured cells. [¹⁸F]ML-10 can be used to detect apoptotic cancer cells (Simpson et al., 2013), and assess the response of brain cancer to therapy (Allen et al., 2012; Oborski et al., 2014). [¹⁸F]ML-10 could be used for apoptosis imaging in murine myocardial infarction model (Fischer et al., 2022 and 2023).

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

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 (immune checkpoint inhibition, ICI). To select appropriate patients for the targeted therapy, the PET radioligands based on therapeutic antibodies, such as atezolizumab, have been developed. Antibodies that bind to a different epitome of PD-L1 than the therapeutic antibody would avoid potential saturation effects and therefore be preferable as PET ligands (Bansal et al., 2021). ⁸⁹Zr-labelled nivolumab could be useful in imaging activated T-cells in tumours and in autoimmune diseases (England et al., 2018).

Adnectins are engineered ∼10 kDa proteins based on human fibronectin (Lipovsek, 2011). Anti-PD-L1 adnectin BMS-986192 has been labelled with ¹⁸F and ⁶⁸Ga (Robu et al., 2021).

Imaging necrosis

Several SPECT radiopharmaceuticals 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 radiopharmaceutical for specific necrosis imaging (Prinsen et al., 2013).

Literature

Bauwens M. In vivo apoptosis imaging using site-specifically ⁶⁸Ga-labeled Annexin V. Methods Mol Biol. 2016; 1419: 17-26. doi: 10.1007/978-1-4939-3581-9_2.

De Saint-Hubert M, Prinsen K, Mortelmans L, Verbruggen A, Mottaghy FM. Molecular imaging of cell death. Methods 2009; 48: 178-187. doi: 10.1016/j.ymeth.2009.03.022.

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. doi: 10.1007/s10495-017-1384-0.

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. doi: 10.2967/jnumed.115.159285.

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.

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. doi: 10.2967/jnumed.109.063917.

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. doi: 10.1039/c6cc03669d.

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. doi: 10.18632/oncotarget.14730.

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. doi: 10.7150/thno.11548.

Tags: Apoptosis, Mitochondria, Hypoxia

Updated at: 2023-04-11
Created at: 2017-09-13
Written by: Vesa Oikonen