Molecular imaging of cell death

Abstract

Apoptosis (programmed cell death) and necrosis (uncontrolled cell death) are two distinct processes of cell death that have been described. Non-invasive molecular imaging of these two processes can have several clinical applications and has various approaches in pre-clinical research. Apoptosis imaging enables a specific and early measurement of response in cancer patients. In case of acute myocardial infarction (AMI) and cerebral stroke the degree of both apoptosis and necrosis is abundant. Imaging of both types of cell death is crucial for diagnosis and could differentiate between “real” and “rescuable” cell damage. In a pre-clinical setting cell death imaging offers the possibility for dynamic study protocols and repeated measurements of cell death in the same animal.

This review provides an overview of the radiopharmaceutical development and in vivo evaluation of apoptosis and necrosis detecting radioligands that have emerged so far. Some apoptosis radiopharmaceuticals have made it to clinical trials (^99mTc-labeled Anx and ¹⁸F-ML-10) while others need further optimization and evaluation (e.g., ¹⁸F-WC-II-89). ^99mTc-glucarate has been widely used in patients to image necrosis, but this radiopharmaceutical only works early after the onset of necrosis. Other necrosis avid probes like ¹²³I labeled hypericin and its monocarboxylic acid derivative and ^99mTc(CO)₃-bis-hydrazide-bis-DTPA pamoic acid need further evaluation but show already promising results for imaging of necrosis.

As a general conclusion molecular imaging of both apoptosis and necrosis is necessary to understand the cell death process in several pathologies.

Introduction

Apoptosis, or programmed cell death, plays an essential role in the development and maintenance of a multicellular organism and contributes to both normal physiology and pathology [1]. This highly regulated and genetically defined cellular process forms the main mechanism by which cells die, both in healthy and diseased tissues. An extrinsic or receptor mediated pathway and an intrinsic or mitochondrial pathway to induce apoptosis have been described [2]. Both pathways lead to the activation of a cascade of cysteine aspartic acid proteases, called caspases. These caspases then cleave functional and structural intracellular proteins. First the structure of the cytoskeleton is modified through proteolysis of different substrates of the cytoskeleton. Typically, apoptosis is characterized by specific changes such as cell shrinking, chromatin condensation, nucleus fragmentation, and morphological changes of cell membrane. The exposure of phosphatidylserine (PS) on the outside of the cell membrane serves as an “eat me” signal for phagocytes [3]. Exposure of phosphatidylethanolamine (PE) on apoptotic cells correlates well with PS exposure in the early phase of apoptosis [4]. Contrary to PS the presence of PE on apoptotic cell surface plays a regulatory role. Blebbing and the formation of apoptotic bodies are essential processes in which intracellular components are discretely packaged and designated to be engulfed by phagocytes without causing inflammation. The transmembrane movement of PE is especially enhanced on blebs of apoptotic cells which attributes to PE-mediated reorganization of actin filaments [5].

Necrosis on the other hand is an accidental, passive and unregulated form of cell death that results from physiochemical damage and sudden metabolic failure. Characteristic for necrosis is swelling of the cell and flocculation of the chromatin. Cell blebs are formed and the integrity of the cell membrane is lost. Water influx and ion redistribution cause the cells to swell and finally disintegrate by which the cell content is released. It is assumed that the release of proteolytic enzymes and other cytosolic material leads to inflammation. Apoptosis and necrosis seem to have different biochemical, morphological and physiological characteristics. However, the two processes of cell death are not so far separated from each other since for example ATP depletion leads to a switch from apoptosis to necrosis [6].

In pathological circumstances two different situations concerning cell death can occur. In some situations apoptosis and necrosis are abundant, as in organ rejection and cardiovascular and neurodegenerative diseases. In other situations the opposite occurs with an insufficient degree of dying of cells as in the case of cancer. Therefore, new therapies are often designed to prevent or induce cell death. Specific radiotracers that can differentiate between apoptosis and necrosis and non-invasively locate and identify cell death at every time point in these pathologies and allow to follow the evolution and determine prognosis could play an important role in medical diagnostics and therapy management.

In oncology molecular imaging of cell death has gained interest for the assessment of tumor response to cancer therapy. Treatment of cancer involves surgery, chemotherapy, radiation therapy, immunotherapy, monoclonal antibody therapy or other methods. The treatment strategy is based on the type of cancer, the location, the grade of the tumor and the stage of the disease, as well as the general condition of the patient. Unfortunately, treatment is not always effective and can have toxic side-effects. Therefore useless, toxic and expensive therapy should be avoided and the most effective treatment should be found for each individual patient. In recent years a lot of new therapies have been developed and currently a number of experimental cancer treatments are being tested in clinical trials. Also combined therapies have become more and more effective. This increases the opportunities for successful treatment of cancer patients and implicates a need for early response assessment to therapy with high quality imaging techniques.

Most current therapy evaluation strategies are based on volumetric and morphological criteria as detected by means of anatomical imaging such as computed tomography (CT) or magnetic resonance imaging (MRI) (e.g., RECIST criteria) [7]. However, morphological changes usually occur 2–3 months after the start or change of treatment and furthermore volume measurements can be hampered by necrotic tissue or scar formation. On the other hand functional molecular imaging by means of positron emission tomography (PET) using ¹⁸F-fluorodeoxyglucose (¹⁸FDG) has been studied the last years as a tool for therapy response assessment revealing highly predictive values already 1–2 weeks after the start of a therapy [8]. However, since ¹⁸FDG is also taken up by inflammatory cells, which are involved in the removal of death tumor cells after therapy, an underestimation of the therapy effect is possible [9]. Another fact that should be taken into account is that some targeted therapies have direct effects on glucose metabolism (e.g., treatment with tyrosine kinase inhibitors (TKI) such as imatinib and gifitinib) inducing glucose-receptor internalization [10]. Some tumor cells can also change their energy source and survive. Therefore, a decreased ¹⁸FDG uptake after treatment with TKI’s does not necessarily represent cell death. As most cancer therapies have a pro-apoptotic effect and resistance hereto can lead to therapy failure [11], [12], molecular imaging of apoptosis offers a specific and early (within 3 days) way to study tumor response to pro-apoptotic cancer therapy [13]. As apoptotic cells can become necrotic and necrosis can also be a result of anti-cancer therapy, molecular imaging of necrosis may be an indicator for the effectiveness of a therapy [14].

In various cardiovascular disorders such as myocardial infarction, myocarditis, cardiotoxicity, cardiomyopathy, cardiac allograft rejection, heart failure and atherosclerosis, cell death – both necrosis and apoptosis – plays an important role in the pathogenesis. Acute myocardial infarction (AMI) is currently being diagnosed with ECG, blood testing and echocardiography. Myocardial perfusion imaging agents such as ^99mTc-tetrofosmin can be used to evaluate the severity, location, and size of the infarct [15]. However, perfusion tracers cannot differentiate between hibernating, stunned, scarred, reversibly and irreversibly injured myocytes. Therefore diagnosis, prognosis and treatment planning for AMI could be more accurate by using specific agents to visualize necrosis and apoptosis. Defining necrosis and apoptosis in AMI could also help in the debate as to when, how and to what extent both forms of cell death contribute to the size of the infarct and subsequently to left ventricular remodeling. All currently available treatment options for AMI aim to reperfuse the infarct related artery. Large clinical trials have shown that early reperfusion therapy, whether mechanical or pharmacological, has a huge clinical benefit on patient outcome in 90–95% of patients. However, despite these successes, up to 40% of the AMI patients later die of heart failure [16], [17], [18]. Therefore, two major complications of AMI are infarct expansion and extension. There is sufficient evidence that in the initial insult of AMI, cardiomyocytes die mainly by necrosis [19], [20]. But there is also evidence that these cells continue to die especially following reperfusion therapy (the so-called reperfusion injury) by both necrosis and apoptosis [21], [22], [23], [24]. Current understanding of the biochemical and molecular processes involved in both forms of cell death may provide new opportunities for therapeutic interventions in the near future [25].

The use of necrosis and apoptosis specific agents could also prove useful to monitor cardiac allograft rejection which is currently monitored by multiple myocardial biopsies and scanning for morphological features [26]. In heart failure muscle cell death is believed to be an important pathological condition. Imaging cell death is an interesting diagnostic tool in these patients and might enable intervention preventing more cardiac function loss [27]. In atherosclerosis it is clinically important to differentiate between stable and unstable plaques which show increased apoptosis levels and could develop thrombosis with cerebrovascular accidents [28].

Finally, detecting neuronal cell death in patients with acute stroke could localize the real cerebral damage, predict outcome and monitor therapy in these patients. There is an immediate irreversible necrosis caused by prolonged ischemia and apoptosis occurring over a period of time in the penumbra of the infarct [29]. The survival of neurons is thought to be particularly important in the penumbra. As apoptosis is a multistep process with the lag phase lasting a significant length of time, it may be possible to block the apoptotic cascade and to save injured neurons [30]. Diffusion and perfusion weighted MRI images have shown to be able to differentiate between the ischemic core and the ischemic penumbra [31]. The ischemic penumbra is a region of incomplete ischemia with maintained cell integrity adjacent to the zone of complete ischemia [32]. This zone gradually expands into the ischemic penumbra and in human studies the penumbra can be found up to 48 h after stroke onset which defines the time window for therapeutic interventions [33]. However, these zones are homogeneous regions defined by perfusion and diffusion deficit and do not necessarily reflect cell death. As a consequence direct imaging of cell death could better identify sites of varying degrees of brain injury on a molecular level. Imaging of necrosis and apoptosis might differentiate effective cerebral damage from “rescuable” cell damage.