Defining the Role of Annexin A5 Imaging in Cancer Treatment and Surveillance

Review

Counting Heads in the War against Cancer: Defining the Role of Annexin A5 Imaging in Cancer Treatment and Surveillance

Maarten F. Corsten,1,2 Leonard Hofstra,1 Jagat Narula,3 and Chris P.M. Reutelingsperger2

Departments of 1Cardiology and 2Biochemistry, Cardiovascular Research Institute Maastricht, University Maastricht, MD Maastricht, the Netherlands and 3Department of Cardiology, University of California, Irvine, School of Medicine, UCI Medical Center, Orange, California

Abstract reliable benchmark for therapy evaluation, the revealed diversity The unveiling of the heterogeneous nature of cell death modes in cell death mechanisms renders its practical feasibility less has compromised the long-lived consensus that cancer straightforward than the theory suggests. Recent findings strongly treatment typically kills cancer cells through apoptosis. indicate that the vast majority of cell death mechanisms can be detected and visualized in vivo with Annexin A5, which senses Moreover, it implies that measures of apoptosis may be misleading indicators of treatment efficacy. Simultaneously, and binds to lipid alterations on the membrane surface of dying it has become clear that phosphatidylserine exposition, cells, irrespective of their dying mechanism (and thus does not traditionally considered a hallmark of apoptosis, is also just recognize apoptosis, as has long been believed). Therefore, associated with most other cell death programs, rendering cell death imaging with Annexin A5 might circumvent the death phosphatidylserine an attractive target for overall cell death mode diversity problem. This review aims to provide an outlay of imaging. Annexin A5 binds with strong affinity to phospha- the developments and arguments that have led to this conclusion. tidylserine and hence offers an interesting opportunity for visualization of aggregate cell death, thus providing a fit Basic Model of Cancer Development benchmark for in vivo monitoring of anticancer treatment. The unraveling of the complex process of cancer development This might be of significant value for pharmacologic therapy has been a key clinical target throughout the last decades. Vast development as well as clinical monitoring of treatment amounts of research have crystallized into a hypothetical basal success. (Cancer Res 2006; 66(3): 1255-60) model of tumorigenesis, in which the impairment of cells’ natural defense against mutations is recognized as a crucial initiator step Introduction (3). This situation is called ‘‘genomic instability’’ and arises when Whereas cancer proceeds to approach the world’s leading cause mutations occur in genes responsible for either DNA maintenance of death position, oncologic medicine is adapting its strategy (caretakers), such as repair genes, or cell cycle government regarding cancer treatment, focusing to a higher extent at (gatekeepers). Gatekeepers represent the organism’s ultimate individual differences in drug responses. The underlying philo- safeguard of tissue homeostasis, due to their ability to induce cell sophy of this personalized medicine approach is to identify what cycle arrest or apoptosis in cells following a range of stresses, drug works best in which patient at what time, to be able to including DNA damage (4). A typical gatekeeper example is the p53 provide personally tailored therapy and hence improve success tumor suppressor protein, which can initiate programmed cell of treatment. Because this strategy requires fast information on death upon detection of genomic stress (5, 6), hence removing the whether or not a drug is working in individual patients, demand for potentially noxious cell from its environment. Its essential more rapid and reliable readouts of treatment efficacy is soaring. importance for preventing cancer development is reflected by Besides improving and speeding up the development of novel the fact that more than half of all human tumors reveal mutations therapies, personalized treatment could benefit patients directly, by in the p53 gene (7). Upon disabling of these caretaker and identifying the optimal therapeutic agent while simultaneously gatekeeper controls, cells become susceptible to the accumulation decreasing side effects suffered from ill-working treatments. of numerous DNA mutations without being repaired or deleted To measure treatment efficacy in vivo more rapidly than with through apoptosis. In contrast to normal cells, these genomically conventional techniques, such as computed tomography or unstable cells are capable of acquiring sufficient mutations to positron emission tomography imaging with glucose analogue develop the key features of malignant cells (8). 18F-fluoro-2-deoxyglucose, other molecular imaging approaches (1) than the latter have received growing attention. Cornerstone Apoptosis in Action: the Cancer Treatment Paradox of these novel approaches is the finding that the degree of It is generally accepted that gaining ‘‘immunity’’ to apoptosis cytoreduction in response to cancer treatment directly correlates represents an essential step in cancer development. Strangely, to both overall disease response to treatment and rate of survival this notion has long coexisted with the prevailing idea that both (2). Although this implies cell death quantification to represent a chemotherapy and radiotherapy kill tumor cells indirectly, by means of damaging DNA and subsequently relying on the cell’s internal apoptotic cascades to finish the job. In fact, numerous studies have used measures of apoptosis to evaluate cancer Requests for reprints: Chris P.M. Reutelingsperger, Department of Biochemistry, Cardiovascular Research Institute Maastricht, P. Debyelaan 25, 6229 AD, Maastricht, the treatment efficacy (9, 10). Moreover, these studies were usually Netherlands. Phone: 31-43-387-5093; Fax: 31-43-387-5104; E-mail: C.Reutelingsperger@ capable of correlating drug-induced apoptotic activity with overall bioch.unimaas.nl. I2006 American Association for Cancer Research. levels of cell death as confirmed with histology as well as with doi:10.1158/0008-5472.CAN-05-3000 clinical responses (11). www.aacrjournals.org 1255 Cancer Res 2006; 66: (3). February 1, 2006

However, under the assumption that cancer cells are resistant to model, in which cell death is regarded a hybrid event on the gliding apoptosis following DNA damage, a logical inference would be scale between two extremes (i.e., apoptosis and necrosis; ref. 24). that normal tissues should be more vulnerable to cancer therapy The precise cell death phenotype is determined by the resultant of than tumor cells. In practice, this seems not to be the case. When cell type, stimulus, and competition of different PCD mechanisms. using in vitro clonogenic survival assays to measure overall cell This model is not without its merits in a sense of providing a new death response to radiotherapy in a variety of normal and paradigm for studying cell death and its modes of appearance. malignant murine tissues, no systematic differences between the Currently, several specific types of cell death have been identified two groups are observed (12). Moreover, tumors with evident in tumors as a result of DNA-damaging agents, as has been defects in the apoptotic pathway should be expected to be more reviewed in more detail by Brown and Attardi (13) and Okada and resistant to these therapies than tumors with intact pathways. After Mak (25). In addition to classic apoptosis, cancer cells may also die evaluating extensively the huge arsenal of available experimental from necrosis, mitotic catastrophe, autophagy, or be functionally and clinical data, Brown and Attardi recently concluded that deleted through what is called senescence (13). Concisely, mitotic at least for nonhematologic cancers, no such pattern could be catastrophe can occur during attempted mitosis following distinguished (13). The discussed data comprise a spread of unsuccessful chromosome segregation, a situation that requires apoptotic deficiencies, including p53 knockouts (14, 15) and malfunctioning of multiple cell cycle checkpoints (26). Catastro- models of overexpression of the proapoptotic protein Bcl-2 (16), phic cells are typically large and nonviable, containing multiple as well as other members of the Bcl-2 family (17). Together, these nuclei with chromosome fragments. Their compromised nuclear findings strongly indicate that the apoptotic capabilities of cells blueprint eventually causes cell death, which may or may not occur have marginal influence on cell survival following DNA damage. through apoptosis, yet if so, it usually does so in a p53-independent This implies that the consensus of cancer therapy typically and manner (27). Autophagy represents genetically regulated self- specifically triggering apoptosis cannot and should not be digestion of the cell. In a caspase- and p53-independent fashion, maintained. organelles are incorporated in autophagosomes, subsequently How then does therapy kill tumor cells? And how can merging with lysosomes to cause content degradation (28). In the described data be synthesized with the occasional success of contrast, senescent cells do not necessarily die, but irreversibly stop apoptosis measuring therapy evaluation assays? To illuminate this dividing, long before their telomere-restricted maximum amount of paradox, we first need to take a closer look at the phenomenon of divisions has been completed (25), resulting in functional deletion. cell death. Consequently, it has been argued that despite of its crucial role in tumorigenesis, apoptosis might play a relatively modest role in cancer response to treatment, because its pathways are assumed The Different Faces of Cell Death to have been compromised. More likely, mitotic catastrophe and The classic dual division of cell death, as originated in the senescence are responsible for the major share of treatment seventies (18), distinguished between necrosis and apoptosis efficacy (29). (programmed cell death or PCD). This model recognized necrosis Concluding, dependent on cell and environmental context, cell as the common insult-induced type of cell death, characterized by death may present in many different forms, rendering the dual cell swelling, membrane rupture, and subsequent cytoplasmatic necrosis/apoptosis model of cell death a shortcoming framework content release in the cell’s direct environment, hence invoking from which to study and evaluate cell killing by antitumor agents. undesirable inflammation. Its stereotypical counterpart, apoptosis, was perceived to embody a highly organized mode of cellular ‘‘suicide,’’ with a group of biological executioner proteases called Evaluating Tumor Treatment Efficacy caspases responsible for its morphologic and biological features The extended model of cell death sheds new light onto the (19). The most obvious are cytoplasmatic shrinkage, chromatin paradoxical findings regarding apoptosis as described earlier. To condensation, DNA degradation, membrane blebbing, subsequent start with, the hybrid nature of cell death makes it conceivable formation of apoptotic bodies, and importantly, no inflammatory that studies (mistakenly) focusing on apoptosis as a measure of response. This dual division, however, has been proven too overall anticancer treatment efficacy will indeed find apoptotic simplistic to maintain. characteristics, in some cases, even quantitatively related to tumor Rather than a sharp boundary, research has unveiled consider- regression. More importantly, however, the model provides theo- able overlap between necrosis and apoptosis. For instance, cells retical backing for the poor correlation between tumor sensitivity undergoing apoptosis (which is an energy-consuming process) to treatment and the condition of apoptotic pathways, because have shown the ability to switch to necrosis upon energy depletion the cell has multiple alternative pathways at its disposal when (20, 21), whereas the opposite may also occur when the noxious apoptosis fails. stimulus driving a cell into necrosis is removed before the end (22). Unfortunately, the corollary of the limited role of apoptosis in Interestingly, it was also shown that PCD-inducing signaling from tumor treatment response is that measures of apoptosis do not the cell’s environment may result in both apoptotic and necrotic necessarily reflect overall cell killing. This could easily lead to phenotypes of cell death, depending on cell type or cellular content wrongful conclusions in cases where tumor response is primarily (23). This not only implies that both modes might be closely effected through mitotic catastrophe or autophagy. This problem intertwined, but moreover, that necrosis can be the result of a was recently elaborated by Brown and Attardi (13). They pointed regulated initiation of cell death, in contrast to the widely perceived out that the use of assays measuring clonogenic potential of tumor notion of necrosis as a passive process following damage. cells after treatment in vitro (or, in short, clonogenic survival In 2001, Leist and Jaa¨ttela¨pointed out that caspases, although assays) leads to much more reliable efficacy evaluation. This is renown as the central executioners of apoptosis, are not always because this method considers the net product of all types of cell involved in PCD mechanisms (24). They suggested a less rigid death as well as growth arrest over a longer time span. In fact,

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2006 American Association for Cancer Research. Counting Heads in the War against Cancer it has been shown that when comparing treatment responses of the cell membranes of apoptotic cells (30–33). As this quality is tumor cells with either intact or compromised apoptotic pathways, hardly affected by labeling with optical or nuclear probes, in vivo assays focusing on the degree of induced apoptosis report lower molecular imaging of cell death using Annexin A5 (Fig. 1) has been sensitivity of the compromised cells, whereas clonogenic survival applied successfully in a variety of (patho)physiologic conditions, assays reveal no difference in long-term net effectiveness between including cardiovascular and oncologic medicine (11, 34–42). the two (13). These results unveil the significance of alternative It has long been assumed that only apoptotic cells externalize death mechanisms, stressing the need for caution while interpret- phosphatidylserine. Accumulating experimental data, however, ing the conclusions of numerous past studies. indicate that phosphatidylserine externalization during cell death The vital shortcoming of clonogenic assessment, however, is that is by no means an exclusive apoptotic event. The first step in irrespective of its reliability, it is not feasible for in vivo imaging. this respect was the finding that activation of caspases is not a For that reason, it cannot incorporate the complex interactions of necessary condition for phosphatidylserine externalization and that the cellular environment and more practically, it does not yield a the latter occurs in nonapoptotic cell death phenotypes just solution for the clinical need for rapid therapy evaluation of patient as well (43–46). Moreover, it has recently been shown by several treatment. In contrast, new insights concerning the membrane groups that even necrotic cell death is associated with active association of phagocyte attractant phosphatidylserine on dying presentation of phosphatidylserine at the cell membrane (47). cells provide an attractive opportunity for noninvasive in vivo For both apoptosis and necrosis, Brouckaert et al. showed that imaging of cell death following antitumor treatment. Moreover, efficiency of phagocytosis is markedly compromised by competitive evidence indicates that this approach might very well circumvent occupation of phosphatidylserine with recombinant Annexin A5, the above described cell death type–related problems. hence reaffirming the functional importance of phosphatidylserine in both cell death modes (26, 48, 49). Additional studies have revealed that regarding phosphatidylserine externalization, mitotic Annexin A5: Detection of the Cell Death Signature catastrophe, and autophagy form no exception (48). Senescent cells, Annexin A5 is an endogenous protein that binds with high logically, do not express phagocyte attractants, because they do affinity and specificity to phosphatidylserine, which is presented on not necessarily die. Nonetheless, their eventual perishing typically

Figure 1. Schematic presentation of the principle underlying cell death imaging with labeled Annexin A5. Living cells actively internalize phosphatidylserine (PS, red dots) to the inner leaflet of their cell membrane. Upon activation of cell death mechanisms, the opposite occurs: phosphatidylserine becomes actively externalized and is presented at the membrane surface to attract phagocytes. Annexin A5 binds with high affinity to externalized phosphatidylserine and can be labeled with molecular tracer groups to allow for in vivo cell death monitoring with a variety of imaging modalities. www.aacrjournals.org 1257 Cancer Res 2006; 66: (3). February 1, 2006

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2006 American Association for Cancer Research. Cancer Research occurs through either apoptosis or mitotic catastrophe, in both Annexin A5 into a very promising candidate to meet the need for rapid cases accompanied by phosphatidylserine externalization (10, 50). in vivo imaging of aggregate cell death responses to tumor treatment. It needs to be stressed that only when used with a second marker showing an intact cell membrane can Annexin A5 be assumed to specifically monitor externalization of phosphatidyl- Annexin A5 Imaging of Treatment Efficacy: Caveats serine. Under controlled laboratory conditions, these conditions and Opportunities can be met. However, when used as a single agent in patients, Important to remind regarding imaging of cancer therapy efficacy interpretation of Annexin A5 is complicated by the ability of the is the fact that aggressive tumors are notorious for displaying protein to bind both externalized as well as internal phosphati- much higher levels of cell turnover than benign processes. This dylserine when the plasma membrane is leaky, as occurs in implies that snapshot imaging of Annexin A5 binding might be necrosis. However, because the latter cause of membrane an inadequate measure of tumor response to treatment, prone to disruption is just as well associated with dying of cells, it can be mistaking tumor aggressiveness for treatment success. Thus, argued that this would not necessarily compromise the final it seems more advisable to base therapy efficacy evaluation upon imaging objective, which is measurement of overall cell death. Annexin A5 uptake compared with baseline images, preferably These novel insights obviously yield important implications measuring signal uptake at multiple time points. Additionally, towards the Annexin A5 imaging protocol. Whereas the bad news is imaging over too short a time period following therapy could entail that Annexin A5–positive results in a variety of studies from the past a pitfall, because cell death programs other than apoptosis might might have mistakenly been attributed to apoptosis alone, the good not become discernible at equally short time horizons. Further news concerning in vivo cancer treatment evaluation is exciting. research will be required to identify optimal imaging timeframes, After all, if phosphatidylserine exposure is a hallmark of the dying cell, which are likely to depend on cancer type and treatment. irrespective of the involved cell death program, then phosphatidyl- Taking the above into account, the use of Annexin A5 imaging serine detection using Annexin A5 overcomes the death mode– for monitoring cancer treatment offers attractive perspectives in related problems as described in earlier sections. This transforms both the pharmaceutical and clinical arena. Regarding the

Figure 2. Example of in vivo evaluation of anticancer drug response with fluorescence molecular tomography using Annexin A5/Cy5.5 in mice. Lewis lung carcinoma cells, sensitive and resistant to cyclophosphamide, were injected into the right and left mammary pads, respectively. A to D, four consecutive tomographic slices with (D) positioned closest to the ventral surface, showing higher tumor response to treatment in sensitive tumor cells. Images reproduced with permission from Vasilis Ntziachristos, Ph.D. (Center for Molecular Imaging Research, Massachusetts General Hospital and Harvard Medical School, Boston). Copyright 2004, National Academy of Sciences.

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Figure 3. Evaluation of the anticancer therapy efficacy in lymphoma patients with single photon emission computed tomography nuclear imaging using 99mTc/Annexin A5. A-D and E-H, total body (nontomographic), transverse, sagittal, and coronal images of a patient before and after treatment, respectively. Images obtained after radiotherapy show clear uptake of 99mTc/Annexin A5, revealing positive cell death response to treatment. Images reproduced with permission from Rick Haas, M.D. (Department of Radiotherapy, The Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital, Amsterdam, the Netherlands). Copyright 2004, Int. J. Radiation Oncology Biol. Phys., Elsevier Publishing. pharmaceutical industry, fast and reliable in vivo evaluation of is currently primarily evaluated through awaiting macroscopic aggregate cell death could accelerate the search for and development regression, often taking weeks, patients bear the risk to suffer from of novel antitumor strategies and compounds. Herein, the combi- possibly ineffective therapy. Thus, faster recognition of treatment nation of Annexin A5 with rapidly improving imaging techniques (in)efficacy should allow for much faster switching to more for small animal (Fig. 2) and clinical imaging can be expected to successful treatment alternatives, significant reduction of unnec- yield significant benefits (11, 51). Moreover, the current attention essary side effects, and consequently marked cost reduction. shift towards personalized medicine and local drug delivery through targeted therapy will likely increase the need for live molecular Outlook approaches of cell death imaging even further in the future. Molecular imaging of apoptosis with Annexin A5 seems to be a Concerning the evaluation of cancer therapy based on Annexin fruitful approach for cell death recognition in vivo. Novel insights A5 uptake in patients, initial results have been very promising reveal that Annexin A5 cell death recognition is not limited to (Fig. 3), although significant additional clinical research is still apoptosis but extends to the vast majority of alternative cell death required. In addition, further improvement of the clearance and mechanisms. Thus, it meets the growing oncologic demand for a biodistribution profile of the radioligand is also recommended and general cell death marker, which has arisen from the discovery 99m feasible. Technetium/HYNIC/Annexin A5 has been used suc- that a wide spread of cell death mechanisms is involved in tumor cessfully in clinical studies measuring cell death in lesions above response to treatment. In the future, the Annexin A5 imaging the diaphragm. The major drawback of this radioligand seems to protocol could contribute substantially to the trend of personal- be its renal retention, which makes it difficult to measure cell izing cancer medicine by providing a rapid readout of treatment death in the abdominal region. Replacing the HYNIC group with an efficacy and hence be of value for both research and development endogenous chelation site for 99mTechnetium decreased renal reten- and clinical monitoring of patient treatment. tion to levels allowing measurement of cell death in the abdomen (ref. 52). The potential gains from this therapy evaluation strategy Acknowledgments involve aspects of time, cost, and side effects. As cancer treatment Received 8/24/2005; revised 11/30/2005; accepted 12/15/2005. www.aacrjournals.org 1259 Cancer Res 2006; 66: (3). February 1, 2006

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2006 American Association for Cancer Research. Counting Heads in the War against Cancer: Defining the Role of Annexin A5 Imaging in Cancer Treatment and Surveillance

Maarten F. Corsten, Leonard Hofstra, Jagat Narula, et al.

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