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Abstract

In this review, data on noninvasive imaging of apoptosis in oncology are reviewed. Imaging data available are presented in order of occurrence in time of enzymatic and morphologic events occurring during apoptosis. Available studies suggest that various radiopharmaceutical probes bear great potential for apoptosis imaging by means of positron emission tomography and single-photon emission computed tomography (SPECT). However, for several of these probes, thorough toxicologic studies are required before they can be applied in clinical studies. Both preclinical and clinical studies support the notion that ^99mTc-hydrazinonicoti-namide-annexin A5 and SPECT allow for noninvasive, repetitive, quantitative apoptosis imaging and for assessing tumor response as early as 24 hours following treatment instigation. Bioluminescence imaging and near-infrared fluorescence imaging have shown great potential in small-animal imaging, but their usefulness for in vivo imaging in humans is limited to structures superficially located in the human body. Although preclinical tumor-based data using high-frequency-ultrasonography (US) are promising, whether or not US will become a routinely clinically useful tool in the assessment of therapy response in oncology remains to be proven. The potential of magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) for imaging late apoptotic processes is currently unclear. Neither ³¹P MRS nor ¹H MRS signals seems to be a unique identifier for apoptosis. Although MRI-measured apparent diffusion coefficients are altered in response to therapies that induce apoptosis, they are also altered by nonapoptotic cell death, including necrosis and mitotic catastrophe. In the future, rapid progress in the field of apoptosis imaging in oncology is expected.

THE CRITERIA USED TO DEFINE objective remission, stabilization, or progression of human malignancies following chemo- and/or radiotherapy are based on morphologic imaging and hence on volumetric changes (computed tomography [CT] or magnetic resonance imaging [MRI] based), with a significant reduction in volume usually ensuing at the earliest 2 to 3 months following effective treatment. During this period, non-responders suffer from unnecessary toxic side effects related to the treatment and at the same time are deprived of other potentially beneficial treatments. Accordingly, methods that allow for a more rapid assessment of response to chemo- and/or radiotherapy in cancer patients are warranted.

Apoptosis is a natural, orderly, and energy-dependent form of programmed cell death that causes cells to die without inducing an inflammatory response. Available preclinical and clinical data support the notion that many of the effects of radiotherapy and chemotherapy are mediated by rapid induction of apoptosis, peaking within the first 24 hours following treatment administration.^1,2 Thus, a noninvasive imaging technique able to monitor baseline and therapy-induced apoptosis in real time could allow for assessment of response to chemotherapy and radiotherapy as early as 24 hours following treatment instigation.

Two main apoptotic pathways have emerged: the mitochondrial pathway (intrinsic pathway) and the death receptor pathway (extrinsic pathway). The intrinsic pathway is mediated by the Bcl-2 superfamily members that interact with the mitochondrial membrane, whereas the extrinsic pathway is governed by the tumor necrosis factor superfamily of ligands and receptors interacting at the cytoplasmic membrane.³ Activation of either pathway implies activation of caspases, cysteine aspartate–specific proteases, which under normal conditions exist as inactive zymogens. Both pathways converge on the key enzyme caspase-3.⁴ Following caspase-3 activation, the morphologic events of apoptosis quickly follow. One of the earliest biochemical events occurring during the apoptotic process is the externalization of phosphatidylserine (PS).⁵ PS serves as a signal for the phagocytes to recognize and engulf the apoptotic bodies, without causing an inflammatory response.⁶ Importantly, exposure of PS is not an exclusive apoptotic hallmark; viable myoblasts, megakaryoblasts, and necrotic cells also present PS at the cell membrane. Following PS externalization, cytoplasm shrinkage and deoxyribonucleic acid (DNA) degradation occur, followed by cell shrinkage, membrane blebbing, and fragmentation of the cell into apoptotic bodies (Figure 1). Degrading necrotic cells have PS on the inside of their cell membranes, which may also be a target for phagocytosis.^7,8

This article reviews and discusses available data on cell death imaging in oncology using the various imaging techniques currently available as a function of the chronology of enzymatic and morphologic events occurring during cell death, as described above (Figure 2). Nuclear imaging techniques including positron emission tomography/single-photon emission computed tomography (PET/SPECT) are described in detail, whereas other imaging techniques are summarized because a detailed description of these techniques is beyond the scope of this review.

Imaging Early Apoptotic Events: Caspase Activation

Radiolabelled Caspase Substrates/Inhibitors

Caspases represent attractive targets because of their central role in the apoptotic pathway as both the death receptor and the mitochondrial pathway eventually converge at the efffector caspases. Until today, the use of radiolabeled caspase substrates/inhibitors has been limited to preclinical studies. No clinical data are yet available.

Prerequisites for imaging of caspase inhibitors include acceptable cell permeability, good solubility, and irreversible inhibition. Various caspase inhibitors have been radiolabeled for the purpose of apoptosis imaging.

Figure 1.

In a viable, nonapoptotic cell, phosphatidylserine (PS), a membrane phospholipid, is normally confined to the inner leaflet of the cell membrane. In an early apoptotic cell, caspases become activated, PS becomes externalized, DNA fragmentizes, and chromatin condensation occurs. After the induction of apoptosis by an apoptotic signal, the apoptosome consisting of cytochrome c, released out of the mitochondria, and procaspase-8 and −9 are formed. Finally, the executioner caspases (caspase-3, −6, −7) are activated and the cell dies. The late apoptotic cell is characterized by cell shrinkage, membrane blebbing, cell fragmentation, and formation of apoptotic bodies.

Benzyloxylcarbonyl-Val-Ala-DL-Asp (O-methyl)-fluoromethyl ketone (Z-VAD-fmk), a pan-caspase inhibitor, was radiolabeled with ¹³¹I. Uptake measurements were performed in Morris hepatoma cells, which showed expression of the herpes simplex virus thymidine kinase (HSV-tk) gene. After treatment with ganciclovir, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assay revealed 1.3 ± 0.3 and 23 ± 1.1% apoptotic cells immediately and 24 hours after therapy. ¹³¹I (Z-VAD-fmk) uptake was found to be twofold elevated immediately after the end of therapy, with no change during the first 4 hours. However, absolute ¹³¹I (Z-VAD-fmk) uptake was low, explained partly by adsorption to the well material, a general issue for peptides at low concentration, and by fetal calf serum interaction of ¹³¹I (Z-VAD-fmk).⁹

Figure 2.

Various imaging techniques currently available as a function of the chronology of enzymatic and morphologic events occurring during apoptosis. Gd = gadolinium; PS = phosphatidylserine; SPIO = supermagnetic iron oxide.

The nonpeptide inhibitors isatin sulfonamide analogues possess nanomolar potency for inhibiting caspase-3 and caspase-7. The nonpeptidyl caspase inhibitor S-(+)-5-[(2-methoxy-methylpyrrolidinyl)sulfonyl] isatin 1 was radiolabeled with ¹⁸F, and biodistribution and small-animal PET showed rapid blood clearance, suggesting a potential role for this inhibitor for the in vivo detection of apoptosis.¹⁰

Similary, the new isatin sulfonamide analogue WC-II-89 was radiolabeled with ¹⁸F. Biodistribution and caspase-3 activation with ¹⁸F-WC-II-89 were subsequently evaluated in a rat model of cycloheximide-induced liver apoptosis.¹¹ Biodistribution studies and microPET revealed a higher uptake of ¹⁸F-WC-II-89 in the liver and spleen of cycloheximide-treated rats when compared to control rats.¹² The effectiveness of two additional isatin analogues, the pyrrolidine analogue ¹¹C-WC-98 and the azetidine analogue ¹⁸F-WC-IV-3, was recently investigated in the same rat model of cycloheximide-induced liver apoptosis. However, both isatin analogues did not discriminate between varying caspase-3 levels in vivo as well as ¹⁸F-WC-II-89.¹³ ¹⁸F-WC-II-89 may represent a better alternative for imaging apoptosis in tissues as it detects only apoptotic and not necrotic cells; however, further preclinical research is needed to investigate its role as a PET radiotracer for imaging apoptosis in cancer patients.

Caspase inhibitors have the intrinsic disadvantage of binding-site saturation. Therefore, caspase substrates that leave the active enzyme site after cleavage were developed. Ten radioiodinated ¹³¹I peptides containing the amino acid sequence Asp-Glu-Val-Asp (DEVD) sequence selective for caspase-3 were synthesized and evaluated for their uptake kinetics using an apoptosis test system. Jurkat cells were treated with staurosporine to induce apoptosis. Two radioiodinated peptides, Tat_49-57-yDEVDG-NH₂ and Tat_57-49-yDEVDG-NH₂, were favorably taken up by apoptotic cells when compared to control cells.¹⁴

Bioluminescence Imaging

A recombinant luciferase reporter molecule has been developed that, when expressed in nonapoptotic mammalian cells, has attenuated levels of reporter activity. However in apoptotic cells, a caspase-3-specific cleavage of the recombinant product occurs, resulting in the restoration of luciferase activity, which can be detected in living animals with bioluminescence imaging (BLI) (Figure 3).

D54 (human glioma) cells that stably express the recombinant luciferase reporter were inoculated subcutaneously in athymic nude mice and were imaged before and after tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) or phosphate-buffered saline (controls) treatment using BLI. Induction of apoptosis using TRAIL resulted in a rapid increase (threefold) in photon counts derived from in vivo tumors compared to controls.¹⁵ A similar reporter construct, ANLucBCLuc, was developed that constitutes a fusion of small interacting peptides, peptides A and B, with the NLuc and CLuc fragments of luciferase with a caspase-3 cleavage site (DEVD) between pepANLuc and pepBCLuc. During apoptosis, caspase-3 cleaves the reporter, enabling separation of ANLuc from pepBCLuc. On the other hand, interaction between peptide A and peptide B restores luciferase activity by NLuc and CLuc complementation (see Figure 3). Treatment of D54 glioma cells and mice carrying D54 tumor xenografts with temozolomide and perifosine resulted in induction of bioluminescence activity, which correlated with caspase-3 activation. Also, combination therapy of temozolomide and irradiation resulted in an increase in bioluminescence activity when compared to monotherapy.¹⁶

Figure 3.

A hybrid recombinant reporter molecule (ER-DEVD-Luc-DEVD-ER) was constructed wherein the activity of a reporter (luciferase) could be silenced by fusion with the estrogen reporter regulatory domain (ER). Inclusion of a protease cleavage site for caspase-3 (DEVD) between two domains allows for caspase-mediated activation of the reporter molecule after separation from the silencing domain (ie, ER). During apoptosis, most cells activate caspase-3, so apoptosis can be detected in a noninvasive way by using bioluminescence imaging. The ANLucBCLuc apoptosis imaging reporter constitutes the split luciferase (NLuc and CLuc) domains fused to the interacting peptides, peptides A and B (PEPA and PEPB), with an intervening caspase-3 cleavage motif (DEVD). In apoptotic cells, the reporter molecule is proteolytically cleaved by caspase-3, which enables the interaction between pepANLuc and pepBCLuc, restoring luciferase activity.

BLI is particularly well suited for imaging small animals, and because most cells selectively activate caspase-3 during apoptosis, this system can be used for imaging of apoptosis.

An overview of the (radio)ligands used for imaging early apoptotic events (caspase activation) is given in Table 1.

Imaging Phospholipid Reorganization: PS Externalization

Annexin A5 SPECT Radionuclides

One of the most successful and widely applied approaches in apoptosis imaging is based on the annexin A5–PS binding. Annexin A5 (36 kDa) is an endogenous human protein and binds in a calcium-dependent manner with nanomolar affinity to PS. PS externalization is one of the earliest events in the apoptotic cascade, does not depend on the apoptosis-inducing agent or cell type, persists until the cell death program is fully completed, and serves as an eat-me signal for phagocytes, which remove the apoptotic fragments. Annexin A5, therefore, is a sensitive marker of the early to intermediate phase of apoptosis. By labeling annexin A5 with fluorescent and radionuclide tags, detection and quantification of apoptosis in vitro and in vivo can be performed. However, annexin A5–based methods are not entirely specific for apoptotic cells, but also necrotic cells are detected at the time when the necrotic cell wall is permeable and PS-binding sites become accessible for annexin A5.

An overview of the (radio)ligands used for imaging phospholipid reorganization (PS externalization) is given in Table 2.

99mTc-HYNIC-Annexin A5

Preclinical data Blankenberg and colleagues derivatized annexin A5 with hydrazinonicotinamide (HYNIC) and coupled it to ^99mTc before intravenous administration in animals.^17,18 Biodistribution studies of ^99mTc-HYNIC-annexin A5 in rats showed rapid clearance from the blood and the highest uptake in kidneys 30 minutes after injection. Anti-Fas antibody injection in BALB/c mice model and cyclophosphamide treatment of a transplanted murine B-cell lymphoma model resulted in a two- to sixfold increase in the uptake of ^99mTc-HYNIC-annexin A5 at sites of apoptosis.^17,19 To determine the optimal point in time for detection of apoptosis in vivo with ^99mTc-HYNIC-annexin A5 after chemotherapy, rats inoculated with allogenic hepatoma cells were treated with a single dose of cyclophosphamide and ^99mTc-HYNIC-annexin A5 was injected in the rats 4, 12, and 20 hours after the treatment.²⁰ The accumulation of ^99mTc-HYNIC-annexin A5 in tumors significantly increased at 20 hours but not at 4 or 12 hours after a single dose of cyclophosphamide, which was consistent with the rate of apoptotic cells determined by TUNEL and caspase-3 immunostaining.²¹

Table 1.

List of Radioligands Imaging Early Apoptotic Events: Caspase Activation

Radioligand	Imaging Modality	Type of Study	Reference
¹³¹I (Z-VAD-fmk)	SPECT	In vitro	9
¹⁸F pyrrolidinylsulfonyl isatin	PET	Biodistribution in nude mice	10
¹⁸F-WC-II-89	PET	Biodistribution in rats + cycloheximide induced liver apoptosis	11, 12
¹¹C-WC-98	PET	Cycloheximide induced liver apoptosis	13
¹⁸F-WC-IV-3	PET	Cycloheximide induced liver apoptosis	13
¹³¹I-DEVD peptides	SPECT	In vitro	14
Recombinant luciferase reporter molecule	BLI	In vitro + D54-bearing nude mice treated with TRAIL	15
ANLucBCLuc	BLI	In vitro + D54-bearing nude mice treated with temozolomide, perifosine and irradiation	16

BLI = bioluminescence imaging; PET = positron emission tomography; SPECT = single-photon emission computed tomography; TRAIL = tumor necrosis factor–related apoptosis-inducing ligand; Z-VAD-fmk = benzyloxylcarbonyl-Val-Ala-DL-Asp (O-methyl)-fluoromethyl ketone.

Methodological Studies in Patients The safety, biodistribution, and dosimetry of ^99mTc-HYNIC-annexin A5 in human volunteers were determined in a phase I study. Highest uptake was observed in the kidneys (30 minutes and 24 hours after injection). The liver and spleen also showed considerable uptake, whereas the gut showed no uptake even at 24 hours after injection (Figure 4). More than 90% of blood activity was cleared, with a short half-life of 25 minutes.²² Several phase I/II studies subsequently evaluated the utility of ^99mTc-HYNIC-annexin A5 for imaging of apoptosis in human patients. In a pilot study by Vermeersch and colleagues, in 18 patients suffering from primary head and neck cancer, ^99mTc-HYNIC-annexin A5 imaging allowed visualization of all primary head and neck tumors identified by CT scan.²³ Involved lymph nodes were, however, not identified. In a comparable setup, 20 patients suffering from primary or recurrent head and neck carcinomas underwent ^99mTc-HYNIC-annexin A5 tomography and subsequent surgical resection of the tumor. Quantitative ^99mTc-HYNIC-annexin A5 tumor uptake correlated well with the number of apoptotic cells assessed by TUNEL assay performed on the tumor slides, if only samples with no or minimal amounts of necrosis were considered.²⁴ Intraobserver, interobserver, and day-to-day reproducibility of quantitative ^99mTc-HYNIC-annexin A5 uptake measurements proved suitable for clinical applications.²⁵ Finally, to determine whether administration of chemotherapy interferes with quantitative uptake of ^99mTc HYNIC-rh-annexin A5 in healthy human tissues 5 to 7 and 40 to 44 hours after treatment initiation, 11 cancer patients were investigated. Five cancer patients received no treatment (control group), and six cancer patients received chemotherapy and underwent ^99mTc-HYNIC-annexin A5 scintigraphy pretreatment as well as 5 to 7 and 40 to 44 hours after treatment initiation. Prior administration of ^99mTc-HYNIC-annexin A5 and chemotherapy did not interfere with quantitative specific uptake in healthy human tissue in the time schedule used.²⁶

Chemotherapy Response Monitoring In a series of 33 patients suffering from various malignant diseases and in a series of 16 patients suffering from non–small cell lung cancer scheduled for platinum-based chemotherapy or concurrent chemoradiation, chemotherapy-induced changes in tumor ^99mTc-HYNIC-annexin A5 uptake calculated as maximum count per pixel and expressed as percentage to baseline value were compared to treatment response determined according to Response Evaluation Criteria in Solid Tumors (RECIST). A significant correlation was found between ^99mTc-HYNIC-annexin A5 metabolic changes and treatment outcome in all studies.^27–29 In a heterogeneous patient population receiving chemotherapy, changes in relative ^99mTc-HYNIC-annexin A5 tumor uptake were evaluated at baseline and at 5 to 7 and 40 to 44 hours after treatment initiation.³⁰ Imaging results were related to clinical outcome, as defined by RECIST.

Table 2.

List of Radioligands Imaging Phospholipid Reorganization: Phosphatidylserine Externalization

Radioligand	Imaging Modality	Type of Study	Reference
^99mTc-HYNIC-Annexin A5	SPECT	Fas-mediated hepatocyte apoptosis in mice + lymphoma-bearing mice receiving cyclophosphamide	17
		In vitro + Fas-mediated hepatocyte apoptosis in mice	18
		Biodistribution in rats + dexamethasone-induced thymic apoptosis in mice	19
		Hepatoma-bearing rats receiving cyclophosphamide	21
		Biodistribution + dosimetry in human volunteers	22
		Head and neck cancer patients	23, 24
		Intra-and interobserver and day-to-day reproducibility in patients	25
		Influence of chemotherapy on biodistribution in patients	26
		Cancer patients receiving chemo- and/or radiotherapy	27
		Lung cancer patients receiving platinum-based chemotherapy	28
		Cancer patients receiving chemo- and/or radiotherapy	29
		Cancer patients receiving chemotherapy	30
		Lymphoma- and sarcoma-bearing mice receiving irradiation	31
		Lymphoma patients receiving irradiation	32
		Head and neck cancer patients receiving chemoradiation	33
^99mTc-MAG₃-Annexin A5	SPECT	Biodistribution in normal mice	34
^99mTc-EC-Annexin A5	SPECT	Breast tumor-bearing mice receiving paclitaxel	35
		Breast cancer patients receiving (chemo)therapy	36
^99mTc-BTAP-Annexin A5	SPECT	Biodistribution in healthy + patients with different malignacies	37, 38
		Lung, breast and lymphoma patients receiving chemotherapy	39, 40
^99mTc-Annexin-128	SPECT	Biodistribution in mice + cycloheximide induced liver apoptosis	41
^99mTc-HYNIC-B1	SPECT	Biodistribution in mice + mice receiving Dexamethasone and anti-Fas antibody	42
^99mTc-HYNIC-cys-Annexin A5	SPECT	Biodistribution in mice + mice receiving anti-Fas antibody	43
		Cycloheximide induced liver apoptosis + castration in rats	44
¹²³I-Annexin A5	SPECT	In vitro	45
		Biodistribution and dosimetry in mice and humans	46
		In vitro + colorectal tumor-bearing mice receiving farnesyltransferase inhibitor therapy	47
		Dexamethasone induced thymic apoptosis in rats	48
¹¹¹In DTPA-PEG-Annexin A5	SPECT	Biodistribution + breast tumor-bearing mice receiving paclitaxel and/or C225	49
¹⁸F - Annexin A5	PET	In vitro	50–52
		Biodistribution + cycloheximide induced liver apoptosis in rats	53
⁶⁴Cu-DOTA-biotin-Sav Annexin A5	i PET	In vitro + breast tumour-bearing mice receiving PDT	54
[¹²⁴I]m-SIB + ¹²⁵I Annexin A5	PET	In vitro	55–56
¹²⁴I Annexin A5	PET	In vitro + mice receiving anti-Fas antibody	57
[¹²⁵I]SIB Annexin A5 + ¹²⁵I Annexin A5	PET	In vitro + fibrosarcoma-bearing mice treated with 5-FU	58
[¹²⁴I]4IB Annexin A5	PET	In vitro + mice receiving anti-Fas antibody	59
¹²⁵I-MBP-Annexin A5	PET	Mice receiving anti-Fas antibody	60
SPIO-C2A	MRI	In vitro + lymphoma-bearing mice	61
IO-Annexin A5	MRI (T2-weighted)	In vitro	62
Gd³⁺-C2A	MRI (T1-weighted)	In vitro + lymphoma-bearing mice receiving ectoposide or cyclophosphamide	63
Biotinylated-C2A-avidin-Gd³⁺	MRI	In vitro	64
Cy5.5-Annexin A5	NIRF	Gliosarcoma-and lung carcinoma-bearing mice receiving cyclophosphamide	65, 68
ABP50-Cy5.5	NIRF	Dexamethasone induced thymic apoptosis in mice + colorectal tumor-bearing mice receiving Apomab	69

BTAP = 4,5-bis(thioacetamido)pentanoyl; EC = ethylenedicysteine; 5-FU = 5- fluorouracil; HYNIC = hydrazinonicotinamide; MAG₃ = mercaptoacetyltriglycine; PDT = photodynamic therapy; SPECT = single-photon emission computed tomography.

Figure 4.

The biodistribution of ^99mTc-HYNIC-annexin A5 in humans: high uptake in the kidneys, less uptake in the liver and spleen, and low uptake in the bone marrow.

Obtained results suggested that sequential ^99mTc-HYNIC-annexin A5 imaging may allow for assessment of response to chemotherapy within 3 days after treatment initiation.

Radiotherapy Response Monitoring A radiation murine tumor model was established to evaluate the value of ^99mTc-HYNIC-annexin A5 imaging in detecting early-phase apoptosis after single-dose irradiation and in predicting tumor response to radiotherapy. The ^99mTc-HYNIC-annexin A5 uptake in E14 lymphoma significantly increased as the radiation dose escalated from 0 to 8 Gy and significantly correlated with the number of TUNEL-positive cells.³¹ To evaluate the potential of ^99mTc-HYNIC-annexin A5 scintigraphy for monitoring radiation-induced apoptotic cell death in humans, patients with follicular lymphoma underwent ^99mTc-HYNIC-annexin A5 imaging before and after the last radiotherapy fraction of the 2 × 2 Gy involved radiotherapy regimen. Fine-needle aspiration cytology was performed on 5 consecutive days to determine the optimal time window for apoptosis detection. The increase in ^99mTc-annexin A5 after 2 × 2 Gy on day 4 was concordant with the presence of apoptotic cells determined by cytology and correlated with clinical outcome.³² In a more recent study, ^99mTc-HYNIC-annexin A5 scintigraphy was performed before and within 48 hours following the first course of cisplatin-based chemoradiation in patients with head and neck squamous cell carcinoma. ^99mTc-HYNIC-annexin A5 scintigraphy showed a radiation dose–dependent uptake in the parotid glands, indicative of early apoptosis during treatment. However, treatment-induced annexin A5 uptake did not predict outcome.³³ This finding suggests that cellular response to radiation is complex and that additional large-scale studies are needed to clarify the role of ^99mTc-HYNIC-annexin A5 scintigraphy in predicting early radiotherapy response (illustrations of ^99mTc-HYNIC-annexin A5 SPECT in human patients are given in Figure 5 and Figure 6).

Figure 5.

A three-dimensional rendered image of the thorax (s = sternum) of a female patient with uptake of ^99mTc-HYNIC-annexin A5 in a primary breast tumor (indicated by a black arrow).

99mTc-MAG3-Annexin A5

In an effort to decrease the high kidney and liver accumulation observed with ^99mTc-HYNIC- annexin A5, annexin A5 was conjugated to mercaptoacetyltriglycine (MAG₃). Biodistribution data in normal mice showed a significant decrease in kidney and liver uptake 1 hour postinjection. However, accumulation in the small intestine was fivefold higher than observed with ^99mTc-HYNIC-annexin A5 at 1 hour postinjection. So, in conclusion, ^99mTc-MAG₃-annexin A5 showed a clear improvement in renal and hepatic clearance; however, owing to the increased radioactivity in the small intestines, imaging of ongoing apoptosis in the lower abdomen might be more difficult compared to^99mTc-HYNIC-annexin A5.³⁴

Figure 6.

A three-dimensional rendered image of the thorax (s = sternum) of a male patient with uptake of ^99mTc-HYNIC-annexin A5 in a squamous tumor cell mass located above the clavicula (indicated by a black arrow).

99mTc-EC-Annexin A5

Another method to prepare ^99mTc-labeled annexin A5 is the conjugation of ethylenedicysteine (EC) to annexin A5 using sulfo-N-hydroxysuccinimide and 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide-HCl as coupling agents. The obtained yield of EC-annexin A5 is approximately 100%.

Preclinical Data Breast tumor–bearing rats were treated with paclitaxel to demonstrate in vivo cell apoptosis induced by chemotherapy. Significantly increased tracer uptake in tumors was observed 3 days after paclitaxel treatment; however, at day 5 posttherapy, tumor uptake was slightly lower compared to that for control animals not receiving therapy.³⁵

Clinical Data A preliminary clinical study of 10 breast cancer patients was performed with ^99mTc-EC-annexin A5 to image tumor cells undergoing apoptosis. Five patients received induction therapy, consisting of paclitaxel in two patients; fluorouracil, doxorubicin, and cyclophosphamide in one patient; and bcl-2 antisense oligonucleotide therapy in the remaining two patients, 16 hours before the imaging session. Whole-body planar images were obtained at 0.5, 2 to 4, and 18 to 24 hours after injection. In nine patients, detectable ^99mTc-EC-annexin A5 uptake corresponded to the area of palpable invasive disease. In patients receiving induction chemotherapy, the mean tumor to background ratio was higher than in those patients who did not receive chemotherapy (2.6 ± 0.5 vs 1.5 ± 0.2). ^99mTc-EC-annexin A5 may therefore be a candidate to evaluate baseline levels of apoptosis, predict the efficacy of therapy based on the detection of treatment-induced apoptosis, and possibly predict disease progression and prognosis.³⁶

99mTc-BTAP-Annexin A5

The pharmacokinetics and imaging properties of ^99mTc-(4,5-bis(thioacetamido)pentanoyl)-annexin A5 (^99mTc-BTAP-annexin A5) were compared to those of ^99mTc (N-1-imino-4 mercaptobutyl)-annexin A5 (^99mTc-i-annexin A5) in healthy volunteers and in patients suffering from different malignancies. ^99mTc-BTAP-annexin A5 proved to be the most stable, with good and reproducible labeling characteristics, and had the advantage of faster clearance from the body and lower radiation burden.^37,38 However, although ^99mTc-BTAP-annexin A5 may be produced with high radiochemical purity, the time-consuming and elaborate preparation makes it less suitable for clinical purposes. Finally, a relationship between ^99mTc-BTAP-annexin A5 tumor uptake and treatment response (24–48 hours after the first course of chemotherapy) in patients with late-stage lung cancer and lymphoma was established by Belhocine and colleagues.^39,40

99mTc-Annexin A5 Mutants

In an effort to develop improved annexin A5–based imaging agents, an endogenous chelation site (Ala-Gly-Gly-Cys-Gly-His) was added to the N-terminus of annexin A5 to create annexin V-128. Comparison of ^99mTc-annexin V-128 with ^99mTc-HYNIC-annexin A5 showed that ^99mTc-annexin V-128 had an 88% lower renal uptake than ^99mTc-HYNIC-annexin A5 at 60 minutes after injection. Mice were treated with the protein synthesis inhibitor cycloheximide to induce apoptosis. A several-fold increase in uptake of ^99mTc-annexin V-128 in the liver and spleen was found, which correlated well with histologic analysis of the degree of apoptosis determined by TUNEL assay.⁴¹

More recently, two different variants of the native annexin A5, called annexin B1 and Cys-annexin A5, were conjugated to ^99mTc-HYNIC and evaluated as novel apoptosis imaging agents.^42,43 Biodistribution studies revealed similar pharmacokinetics of ^99mTc-HYNIC-B1 in mice when compared to ^99mTc-HYNIC-annexin A5, high renal uptake, and no bowel excretion. Thymus uptake and hepatic uptake of ^99mTc-HYNIC-B1 increased significantly after dexamethasone induction and Fas-mediated induction in mice, respectively.⁴² The second-generation annexin A5, Cys-Annexin A5, was developed by incorporation of a cysteine residue into annexin A5 using site-specific mutagenesis, which allows site-specific conjugation of the HYNIC moiety and subsequent radiolabeling without interfering with the annexin A5–PS interaction. Biodistribution studies of ^99mTc-HYNIC-Cys-annexin A5 in normal mice showed a pattern similar to that of the first-generation ^99mTc-HYNIC-annexin A5 conjugates. There was a 257% increase in hepatic uptake of ^99mTc-HYNIC-Cys-annexin A5 in a murine model of apoptosis when compared to control mice, indicating that ^99mTc-HYNIC-Cys-annexin A5 may yield similar apoptosis imaging results when compared to the first-generation ^99mTc-HYNIC-annexin A5.⁴³ To investigate the utility of the ^99mTc-HYNIC-Cys-annexin A5 for the noninvasive detection of cell death in liver, spleen, and prostate, rats were treated with cycloheximide to induce cell death in the liver and spleen, whereas apoptosis in the prostate was induced by castration. Two hours following cycloheximide administration, a twofold and a fourfold increased accumulation of ^99mTc-HYNIC-Cys-annexin A5 in the liver and spleen, respectively, over controls was observed, which correlated well with TUNEL-positive cells in the tissue sections. Despite the high levels of TUNEL-positive prostate epithelial cells following castration, no increase in ^99mTc-HYNIC-Cys-annexin A5 could be seen. A possible explanation for the latter is the tighter organization of the capillary endothelial cells within the prostate, with the main apoptotic cell types being located behind this barrier. Moreover, a layer of smooth muscle cells, fibroblasts, and connective tissue provides an additional barrier between the target prostate cells and the circulation.⁴⁴

123I-Annexin A5

Preclinical Data Binding experiments with blood platelets demonstrated that ¹²³I-annexin A5 binding was time and Ca²⁺ dependent and indicated that the radiotracer retained its biologic activity.⁴⁵ The biodistribution and dosimetry of ¹²³I-rh-annexin A5 was evaluated in mice. ¹²³I-rh-annexin A5 accumulated primarily in the liver, kidney, stomach, and lung. Clearance was predominantly urinary.⁴⁶ In vitro, the binding of ¹²³I-annexin A5 to tipifarnib (a selective farnesyltransferase inhibitor and anti-tumor agent) –treated cells proved to be significantly higher than binding to vehicle-treated cells. In vivo, LoVo-inoculated athymic mice showed a 40% increase in ¹²³I-annexin A5 uptake 8 hours after a single oral administration of tipifarnib as well as after 3 days of twice-daily treatments with the same dose. Ex vivo TUNEL assays confirmed the presence of apoptotic cells in both in vitro and in vivo experiments.⁴⁷ In an animal model of dexamethasone-induced apoptosis in the thymus, the degree of apoptosis was determined in the same animal at 6 and 11 hours after a single administration of dexamethasone by ¹²³I-annexin A5 and by in vitro evaluation of the apoptotic index in the form of DNA fragmentation on tissue sections using in situ oligo ligation. ¹²³I-annexin A5 uptake significantly increased in the thymus of rats 6 hours after dexamethasone administration and significantly decreased afterward. On the other hand, in situ oligo ligation demonstrated an increase in the apoptotic index at 6- and 11 -hour time points.⁴⁸

Clinical Data In humans, extensive bowel activity and rapid in vivo dehalogenation excluded the use of ¹²³I-rh-annexin A5 for imaging apoptosis in a clinical setting.⁴⁶

111In DTPA-PEG-Annexin A5

Annexin A5 has also been labeled with ¹¹¹In, which has a longer physical life (2.81 days) than ^99mTc, making it possible to detect therapy-induced apoptosis over a prolonged period. Given that apoptosis is a dynamic process and apoptotic cells are engulfed by neighboring cells within hours, it has been suggested that increasing the interval between radiotracer injection and imaging would allow detection of apoptotic cells over a longer period in time, enhancing detection sensitivity.

Both polyethylene glycol (PEG) and diethylenetriamine pentaacetic acid (DTPA) were bound to annexin A5 and ovalbumin. PEG-modified proteins have been shown to exhibit reduced liver uptake and increased blood circulation half-lives. Mice bearing inoculated human mammary MDA-MB-468 tumors were treated with poly(l-glutamic acid)-paclitaxel, monoclonal antibody C225, or a combination of both. Images were acquired 48 hours after the injection of the radiotracer. ¹¹¹In DTPA-PEG-annexin A5 increased in the gastrointestinal tract as well as in the tumors of mice treated with either poly(l-glutamic acid)-paclitaxel or combined poly(l-glutamic acid)-paclitaxel and C225 during 4 days compared to radioactivity in the tumors of untreated mice. There was a significant correlation between tumor uptake of ¹¹¹In DTPA-PEG-annexin A5 and apoptotic index determined by TUNEL assay (r = .87, p = .02). Imaging with ¹¹¹In DTPA-annexin A5 failed to reveal tumors in either untreated mice or treated mice. Intratumoral distribution of ¹¹¹In DTPA-PEG-annexin A5 showed a main distribution in the central zone of the tumors, whereas ¹¹¹In DTPA-annexin A5 was largely confined to the tumor periphery.⁴⁹ No human study has been performed.

Annexin A5 PET Radionuclides

When compared to SPECT, PET offers higher resolution (spatial resolution is about two times better) and higher sensitivity and is more quantitative than SPECT. Additionally, PET accurately corrects for attenuation of photons emitted from the organ of interest, making PET images more accurate in reflecting the true activity of the tracer in the tissue. The disadvantages of PET include the higher cost and the necessity of a particle accelerator (cyclotron) for production of radionuclides such as ¹⁸F.

18F-Annexin A5

¹⁸F has been conjugated to annexin A5 via the compound N-succinimidyl-4[¹⁸F]-fluorobenzoate, [¹⁸F]FSB. [¹⁸F]FSB was subsequently shown to bind to apoptotic cancer cells in a fashion identical to that of fluorescein isothiocyanate (FITC)-annexin A5.⁵⁰ Stimulation of cancer cells with the chemotherapeutic agent etoposide for 6 hours showed an 88% increase in the binding of [¹⁸F]FSB when compared to control cells.⁵¹ Also, apoptotic Jurkat T cells (apoptosis induced by ultraviolet irradiation) showed a 60% increase in ¹⁸F-annexin A5 compared to uptake in nonirradiated Jurkat T cells.⁵² Pretreatment of rats with cycloheximide resulted in a three-to ninefold increase in ¹⁸F-annexin A5 in the livers of treated rats at 2 hours compared to those of control animals. Morphologic analysis and TUNEL assay confirmed the apoptotic levels. Biodistribution studies of normal rats showed the highest uptake of ¹⁸F-annexin A5 in the urinary bladder and kidney. After the bladder and kidney, organs with the highest uptake were the lungs, liver, and spleen.⁵³ However, owing to the short half-life of the isotope (110 minutes), relatively high starting amounts of radioactivity are required to obtain good images. The labeling method is also not yet fully standardized, so more research is required for this PET tracer to be used in patients.

64Cu-DOTA-Biotin-Streptavidin Annexin A5

In tumor-bearing mice, tumor apoptosis after photodynamic therapy (PDT) was visualized by PET using ⁶⁴Cu-(1,4,7,10 tetraazacyclododecane-1,4,7,10-tetraacetic acid) (DOTA)-biotin-streptavidin, following pretargeting of apoptotic cells with biotinylated annexin A5. An avidin chase, after administration of the biotinylated annexin A5, was performed to eliminate free biotinylated products, thus increasing detection specificity and reducing blood background. PDT induces apoptosis by using phthalocyanine dyes as photosensitizers and red light. These dyes have been shown to act directly on tumor cells, causing rapid and extensive damage within the illuminated area. PET scans were recorded up to 13 hours after PDT and delineated apoptosis in the treated tumor as early as 30 minutes after tracer injection. A clear contrast between the treated tumor and the surrounding tissue was reached at 7 hours post-PDT.⁵⁴

124I-Annexin A5

Annexin A5 has also been radiolabeled directly and indirectly with the positron emitter ¹²⁴I (half-life = 4.18 days, E_maxβ⁺ = 1.53, 2.14 MeV). Indirect labeling has been performed by the Bolton-Hunter method, by the prelabeled reagent N-succinimidyl 3-[¹²⁴I]iodobenzoate ([¹²⁴I]m-SIB), and by using [¹²⁴I]N-hydroxysuccinimydil-4-iodobenzoate prepared by iodostannylation of N-hydroxysuccinimidyl 4-(tributylstannyl)benzoate (creating [¹²⁴I]4IB).⁵⁵

Glaser and colleagues iodinated annexin A5 directly by the chloramine-T method to produce ¹²⁴I-annexin A5 and indirectly by the prelabeled reagent [¹²⁴I]m-SIB.⁵⁶ In vitro studies using human leukemic HL60 cells stimulated with camptothecin showed a nonsignificant increase of 17% in directly labeled ¹²⁵I-annexin A5 and a significant 21% increase in [¹²⁵I]m-SIB, suggesting a reduced biologic activity of the directly labeled annexin A5.⁵⁶ Jurkat cells treated with the anticancer drug camptothecin had a 7.5-fold higher ¹²⁴I-annexin A5 uptake compared to untreated cells. A good correlation was found between radioactivity per cell and FITC-annexin A5 binding at different apoptosis levels (r = .93, p < .0001).⁵⁷ RIF-1 cells treated with 5-fluorouracil (5-FU) for 24 hours to induce apoptosis showed higher binding of ¹²⁵I-annexin A5 and [¹²⁵I]SIB-annexin A5 compared to control cells. The extent of binding was dependent on the concentration of 5-FU added.⁵⁸ Administration of anti-Fas antibody to BDF-1 mice resulted in significantly higher ¹²⁴I-annexin A5 hepatic uptake compared to controls at 1 and 2 hours. PET images obtained at 2 hours after injection of ¹²⁴I-annexin A5 in untreated and anti-Fas-treated mice revealed accumulation of the tracer in bladder and stomach in untreated mice, whereas treated animals had massive accumulation in the livers. Also, in vivo ¹²⁴I-annexin A5 uptake derived from PET images correlated with histologically derived apoptotic density (r = .67, p < .001).⁵⁷ The same research group investigated imaging of apoptosis in vivo with [¹²⁴I]4IB-annexin A5 made by using [¹²⁴I]N-hydroxysuccinimydil-4-iodobenzoate prepared by iodostannylation of N-hydroxysuccinimidyl 4-(tributylstannyl)benzoate. In mice treated with anti-Fas antibody, [¹²⁴I]4IB-annexin A5 accumulated in the liver; however, the difference in uptake between untreated and treated animals was not as clear as that previously demonstrated with directly labeled ¹²⁴I-annexin A5.⁵⁹

Collingridge and colleagues also synthesized ¹²⁴I-annexin A5 and [¹²⁴I]SIB-annexin A5.⁵⁸ The uptake of [¹²⁵I]SIB-annexin A5 by RIF-1 tumors growing in mice increased by 2.3-fold at 48 hours after a single injection of 5-FU compared to a 4.4-fold increase in TUNEL-positive cells. Uptake of the directly labeled ¹²⁵I-annexin A5 did not significantly change in 5-FU-treated mice compared to control mice.

In 2005, ¹²⁴I-maltose-binding protein (MBP)-annexin A5 was developed and evaluated in vivo.⁶⁰ Iodinated MBP and albumin were used as control proteins. Treatment with anti-Fas antibody prior to radiotracer injection clearly showed apoptotic hepatocytes by PET. Liver uptake of ¹²⁵I-MBP-annexin A5 was nine times greater for mice treated with anti-Fas antibody than for untreated control animals, which was confirmed when ¹²⁴I-MBP-annexin A5 was used as a radiotracer. Immunohistochemistry on liver sections taken from animals treated with anti-Fas antibody confirmed the presence of apoptotic cells.

Magnetic Resonance Imaging

Zao and colleagues labeled the first C2 domain of the protein synaptogamin I (C2A) with superparamagnetic iron oxide (SPIO) particles that serve as an MRI contrast agent.⁶⁰ Synaptogamin I is a PS-binding protein like annexin A5 but much smaller (14 kDa). SPIO-labeled C2A was shown to bind to the plasma membrane of EL4 and CHO-K1 cells, induced to undergo apoptosis, and in vivo when administered intravenously into drug-treated mice bearing EL4 tumors.⁶¹

Schellenberger and colleagues cross-linked iron oxide nanoparticles with annexin A5 and demonstrated a dose-dependent signal intensity reduction in T₂-weighted images of camptothecin-treated apoptotic Jurkat cells versus untreated controls in vitro.⁶² As SPIO particles are detected by their effects on T₂*, resulting in negative contrast, using MRI it is difficult to discern areas of apoptosis from other hypointensity regions in the tumor. In addition, SPIO particles are relatively large, which can limit access to and clearance from the tumor interstitium. Ionic gadolinium (Gd³⁺) contrast agents are both smaller and give increased signal intensities on T₁-weighted MRI, making them easier to detect. In a recent study by Krishnan and colleagues, the affinity of Gd³⁺-C2A synaptogamin I for PS was demonstrated, and Gd³⁺-C2A synaptogamin I bound to apoptotic and necrotic cells but not to viable cells in vitro.⁶³ In vivo, EL4 tumor–bearing mice treated with a single intraperitoneal injection of etoposide or cyclophosphamide showed a greater accumulation of the PS-active contrast agent compared to control animals 24 hours after the injection of the PS-active contrast agent. Importantly, the magnetic field used in the study was much higher than the strength used in clinical applications, limiting the clinical relevance of the obtained results. Finally, Neves and colleagues used two biotinylated C2A domains of the protein synaptogamin I, which were complexed with a single avidin molecule labeled with gadolinium chelates.⁶⁴ MRI allowed detection of the apoptotic tumor cells in vitro. Low sensitivity of the MRI technique can be improved by contrast agents that give signal amplification. In this way, in vivo imaging of apoptosis in the clinical setting may occur in a sufficient and specific manner.

NIRF Imaging

As opposed to the green fluorescent reporter FITC, far-red and near-infrared fluorescent probes bear high tissue and blood transmittance and show low interference of the scattered light used for exciting fluorescence. Accordingly annexin A5 has been covalently conjugated with the near-infrared indocyanine fluorescent dye Cy5.5 (Cy5.5-annexin A5). Cy5.5-annexin A5 was used to image a 9L gliosarcoma constitutively expressing green fluorescent protein (GFP), and the CR8 variant of Lewis lung carcinoma (LLC) stably transfected with DsRed2 (a variant of the original red fluorescent protein, with improved solubility). The expression of GFP and DsRed2 provides an indication of tumor margins and thus a means of defining tumor-associated NIRF signal intensity. In both tumor models, active Cy5.5-annexin A5 tumor NIRF signal increased two to three times after cyclophosphamide treatment. In the 9L gliosarcoma model, active Cy5.5-annexin A5 bound to tumor cells and endothelial cells.⁶⁵ In a similar study performed by the same research group, an elevated tumor Cy5.5-annexin A5 ratio (tumor NIRF to background NIRF) was found in cyclophosphamide-chemosensitive LLC inoculated mice treated with cyclophosphamide compared to untreated animals, which correlated well with the number of apoptotic cells analyzed by TUNEL assay.⁶⁶

Finally, a 10-fold increase in fluorochrome concentration in cyclophosphamide-sensitive (CCL) and a 7-fold increase in cyclophosphamide resistant (CR-CLL) tumors compared to control studies was observed, which was consistent with the higher apoptotic index confirmed by TUNEL staining and histology.⁶⁷ These results were confirmed by Choi and colleagues 1 year later.⁶⁸ Recently, Cy5.5-labeled activity-based probes (ABPs) that covalently label active caspases in vivo have been developed and were tested in the thymi of mice treated with dexamethasone as well as in tumor-bearing mice treated with the apoptosis-inducing monoclonal antibody Apomab. A fluorescent probe (ABP50-Cy5.5) was developed with high selectivity toward caspase-3 and legumain and with virtually no detectible cathepsin B binding. Cross-reactivity with cathepsins is an important disadvantage of most caspase-based probes because of high constitutive expression of these cysteine proteases in liver, kidney, and spleen. In mice treated with dexamethasone, caspase-3 activity in the thymi was observable at 6 hours after treatment, peaking at 12 hours and then sharply dropping to background levels at 24 hours after injection. In the Apomab-treated mice, maximum caspase activity occurred 12 hours after Apomab treatment, as indicated by total fluorescent signals in the tumors, which closely mirrored levels of caspase-3 and caspase-7 measured by gel analysis. So these authors concluded that direct, noninvasive in vivo imaging of the kinetics of apoptosis has great value for clinical application, and the ABP can be used for this purpose.⁶⁹ However, the disadvantages inherent in planar imaging include lack of quantification, low penetration depth (< 1 cm), and an inability to accurately provide depth and size information.

Imaging Midterm to Late Apoptotic Events: DNA Fragmentation and Chromatin Condensation

An alternative detection method that allows real-time monitoring of apoptosis is high-frequency ultrasound imaging (HFUI, 20–40 MHz), based on acoustic waves. HFUI probes midterm to late apoptotic events, respectively, DNA fragmentation and chromatin condensation, which yield a six times higher ultrasound scatter when compared to nonapoptotic cells. Tunis and colleagues used HFUI (20 MHz) and demonstrated that this method can be used to monitor structural changes within a very low percentage of treated cells in a population, raising the possibility of using this technique in vivo for the monitoring of tumor response.⁷⁰ To determine whether HFUI could be used to detect apoptosis ex vivo, PDT-treated brain tissue was examined and compared to the nonirradiated contralateral side of an unfixed freshly excised brain. Backscatter amplitude measurements demonstrated an increase in ultrasound backscatter at 1.5, 3, and 24 hours in post-PDT brain areas compared to the controls. In an in vivo model, rats with photosensitized areas of their skin exposed to activating light (0, 8.5, and 17 J cm⁻²) were evaluated and an increase in ultrasound backscatter in the epidermis was observed that corresponded to the dose of activating light.⁷¹ More recently, xenografted malignant melanoma tumor response to PDT was evaluated, and a time-dependent increase in ultrasound backscatter after treatment was observed. The increase in ultrasound backscatter correlated well with morphologic results, indicating an increase in apoptotic cell death, with a peak at 24 hours after PDT.⁷² HFUI may be a highly effective and inexpensive tool for anatomic imaging; however, the inability to investigate deeply located tumor lesions and the inability to discriminate necrotic from apoptotic cells seem to make HFUI less appropriate for successful imaging of in vivo apoptosis.

Imaging Midterm to Late Apoptotic Events: Membrane Phospholipid Metabolism

Magnetic Resonance Spectroscopy

1H Magnetic Resonance Spectroscopy

The changes in the lipid structure and fluidity of the cell membrane that take place during apoptosis generate a number of small molecules (cytoplasmic lipid bodies, choline metabolites) that can be directly monitored by water-suppressed lipid ^lH MRS. Blankenberg and colleagues observed signals from mobile intracellular lipids and demonstrated a correlation between the intensity ratio of the 1.3 ppm CH₂- (methylene) and 0.9 ppm CH₃- (methyl) resonance and the fraction of apoptotic cells following induction of apoptosis by doxorubicin in Jurkat T cells.⁷³ This technique was also used to detect apoptosis in vivo in BT4C rat gliomas undergoing ganciclovir–thymidine kinase gene therapy–induced apoptosis and in a murine lymphoma in vivo model treated with chemotherapeutic drugs.^74,75

Contrary to the findings by Blankenberg and colleagues,⁷³ Valonen and colleagues found a late-occurring decrease in the CH₂/CH₃ signal instead of the expected increase in the CH₂/CH₃ signal in apoptotic brain tumors derived from HSV-tk-transfected BT2C glioma cells.⁷⁶ Thus, ¹H magnetic resonance signals from cytoplasmic lipids do not seem to be unique identifiers for apoptosis. To date, it is not known which factors contribute to the increase in the concentration of lipids. Also, it will be difficult to eliminate lipid droplet signals from the other sources in the surrounding tissue of the tumor cells. Further studies aimed at clarifying these issues are awaited.

31P Magnetic Resonance Spectroscopy

Many metabolites have been investigated as useful metabolic markers for apoptosis in vivo using ³¹P MRS. Only two metabolites, fructose 1-6-biphosphate (FBP) and cytidine diphosphocholine (CDP-choline), were found to selectively increase in apoptotic cells. FBP accumulates in the apoptotic cell owing to the accompanying activation of the poly-ADP-ribose polymerase (PARP), which uses oxidized nicotinamide adenine dinucleotide (NAD⁺) as a substrate. As a consequence of the depletion of NAD⁺, the glycolytic pathway becomes blocked and an accumulation of the upstream intermediate of the enzyme glyceraldehyde-3-phosphate, including FBP, occurs. Accumulation of CDP-choline in apoptotic cells occurs owing to the acidification that occurs early in the apoptotic process, which results in the inhibition of the enzyme cholinephosphotranferase and in the subsequent accumulation of its substrate, CDP-choline.⁷⁷ ³¹P MRS measurements are relatively insensitive and lack temporal and spatial resolution as the signal owing to FBP was masked by the resonance of other phosphomonoesters. Moreover, it is unlikely that ³¹P MRS will be able to detect apoptosis in the tumor when the percentage of the apoptotic cells is relatively low,⁷⁸ which limits the use of these metabolites clinically.

13C Magnetic Resonance Spectroscopy

Recently, the introduction of dynamic nuclear polarization, which increases sensitivity in ¹³C MRS experiments by 10,000–fold, has allowed the detection of ¹³C labeled substrates in vivo. Lymphoma-bearing mice injected with hyperpolarized (1-¹³C) pyruvate showed a decrease in the flux of hyperpolarized ¹³C label between pyruvate and lactate in the reaction catalyzed by the lactate dehydrogenase after drug-induced (etoposide) cell death.⁷⁹

In general, MRS is significantly limited for in vivo imaging of apoptosis in patients by relatively low sensitivity and poor temporal and spatial resolution.

Imaging Late Apoptotic Events: Cell Shrinkage and Membrane Blebbing

Magnetic Resonance Imaging

1H Magnetic Resonance Imaging

Diffusion-weighted magnetic resonance imaging (DW-MRI) generates image contrast by using the diffusion properties of water within the tissue. Morphologic features associated with apoptosis, including cell shrinkage and membrane blebbing, induce changes in the apparent diffusion coefficient (ADC), which can be quantitatively measured.⁸⁰ The increase in water diffusion caused by apoptosis or necrosis occurs within 2 to 4 days, prior to visible changes in tumor morphology or size. An increase in ADC has been demonstrated in different animal tumor models in response to different kinds of apoptosis-inducing agents.^81,82 In a recent study, ¹H DW-MRI was used to assess early treatment response of death receptor 4 antibody TRA-8 in a mouse breast cancer model. ADCs were calculated and compared to tumor volume and living tumor cell changes. The ADC increase at day 3 was dependent on TRA-8 dose level and was significantly higher in the group receiving the highest dose of TRA-8 compared to the other groups, which received lower doses. The dose-dependent ADC increases were linearly proportional to the number of apoptotic cells and cleaved caspase-3 densities. The therapeutic efficiency of TRA-8 could be detected as early as 3 days by using ADC quantification, before changes in tumor dimension occurred.⁸³ However, ¹H MRI–measured ADC is also altered in response to therapies that induce nonapoptotic cell death, including necrosis and mitotic catastrophe, and an increase in ADC can occur owing to these two ways of cell death in the absence of apoptosis. Therefore, changes in ADC measure the global cytotoxic response to chemotherapy and not just the apoptotic response to treatment.⁸⁴

23Na Magnetic Resonance Imaging

Weighted sodium MRI has also been evaluated to monitor rapid in vivo chemotherapeutic response. Because of the biologic importance of sodium and its relative abundance, sodium MRI is an interesting tool for the study of many pathophysiologic processes. In nude mice inoculated with human prostate cancer cells and treated with antineoplastic drugs (docetaxel or etoposide), a 36% ± 4% increase in weighted sodium images was observed 24 hours after administration of the therapy. Flow cytometry revealed that the elevation of intracellular sodium preceded cell death by apoptosis.⁸⁵ In a more recent study, using the same tumor model, docetaxel induced an increase in intracellular sodium MRI signal (of 30%) with decreased tumor size and an increase in fluorodeoxyglucose uptake compared to control tumors after 24 hours.⁸⁶

Relaxation (T1/T2) MRI

Sierra and colleagues investigated the possible use of longitudinal and transverse relaxations in the rotating frame (T_1p and T_2p, respectively) in rats bearing BT4C gliomas treated with HSV-tk gene therapy to induce apoptotic cell death.⁸⁷ These nuclear magnetic resonance tools were able to discriminate different treatment responses in tumor tissue from day 4 onward, and T_1p and T_2p appeared to be excellent tools to quantify early changes in water dynamics, reflecting treatment response during gene therapy.

In comparison with MRS, ¹H MRI provides much better spatial and temporal resolution, along with very high sensitivity, and is therefore routinely used in clinical applications to measure volume reduction of a tumor. However, it may be difficult to distinguish cell shrinkage, necrosis, and other types of cell death that occur with cancer treatment.

Novel Apoptosis Tracers

Novel probes are being explored for their potency to image apoptosis in vivo, such as the ApoSense family, dansylhydrazone (DNFSH), and small peptides.

The ApoSense family, comprising low-molecular-weight apoptosis markers, is specifically designed for the imaging of cell death in vivo. The compounds do not cross the plasma membrane of an intact viable cell but selectively pass through the membrane and accumulate within the cytoplasm of apoptotic cells from the early stage of the apoptotic process. NST-732, a member of the ApoSense family, demonstrated selective and rapid accumulation within various cell types undergoing apoptosis. In vivo, lymphoma-bearing mice were subjected to radiotherapy and fluorescent NST-732 was intravenously administered. On induction of cell death by irradiation, a drastic increase in NST-732 uptake occurred. TUNEL staining showed that uptake of NST-732 and positive TUNEL staining were colocalized, confirming that the cells labeled with fluorescent NST-732 were indeed undergoing apoptosis.⁸⁸ A novel ApoSense family member, ML-9 (butyl-2-methylmalonic acid), based on an alkyl-malonate motif, was labeled with tritium (³H-ML-9) and was tested in vitro and in vivo. The tumors of control animals manifested low uptake levels of ³H-ML-9, whereas doxorubicin treatment caused a dramatic increase of 18-fold in tumor uptake levels of the tracer. The detection of three distinct hallmarks of apoptosis (DNA fragmentation, caspase-3 activation, and phospholipid scrambling) in the tumors in response to chemotherapy confirmed the association between the increased ³H-ML-9 uptake and chemotherapy-induced tumor cell apoptosis.⁸⁹ A promising ApoSense PET tracer for apoptosis is ¹⁸F-ML-10 (5-fluoropentyl-2-methylmalonic acid). It was first tested in small-animal PET for imaging of neurovascular cell death in an experimental cerebral stroke model. This radiotracer seemed to selectively detect apoptotic but not necrotic cells, and selective uptake was observed in the regions of interest of the infarct.⁹⁰ ¹⁸F-ML-10 has proceeded to phase I/II clinical trials for molecular imaging of apoptosis, including one for the early assessment of response of brain metastases to radiotherapy.⁹¹

DNFSH was developed and evaluated as a small molecule–based tracer for the in vivo imaging of apoptosis. ¹⁸F-DNFSH accumulates in the cytoplasm of apoptotic cells, whereas Alexa 488–labeled annexin A5 is bound on the outside of the cell membrane. Because of the promising in vitro results, ¹⁸F-labeled DNFSH was prepared and radiochemical yields of 50 to 60% were obtained.⁹²

To discover PS-binding peptides that can be used to image apoptosis, an M13 phage display peptide library was screened onto PS-coated enzyme-linked immunosorbent assay (ELISA) plates. The phage with the highest affinity-bound PS in ELISA carried the peptide TLVSSL.⁹³ Another peptide, with sequence CLSYYPSYC, was discovered, and tumor-bearing mice treated with a single dose of an anticancer drug (camptothecin) and fluorescein-labeled CLSYYPSYC peptide showed peptide homing to the tumor. This peptide represents a novel PS-recognizing agent and may be used in the future as a radionuclide or fluorophore probe to detect apoptosis in vivo.⁹⁴ The advantages of the novel probes (NST-732, ³H-ML-9, ¹⁸F-ML-10, and ¹⁸F-DNFSH) include the low molecular weight, which may be characterized by more favorable biodistribution and clearance. In addition, intracellular accumulation will provide higher signal to noise ratios compared to the annexin A5 probes, which bind to the periphery of apoptotic cells. However, the exact mechanism by which these probes targets the apoptotic cell membrane is not known, and high levels of injected material are needed, so further investigation is needed before some of the novel Apoptosis tracers become applicable in clinical trials.

Discussion

Various imaging techniques allow for noninvasive in vivo imaging of apoptosis: PET and SPECT, BLI, NIRF, and MRI.

PET and SPECT represent probably the most sensitive methods for molecular imaging of apoptosis. A broad range of clinical apoptosis-targeting probes are currently available: radiolabeled caspases, annexin A5, and, more recently, ¹⁸F-ML-10 and duramycin. Radiolabeled caspase inhibitors/substrates have been developed for PET and SPECT apoptosis imaging because of their central role in the early phase of the apoptotic process. Available in vitro and preclinical studies suggest that these radiopharmaceuticals bear great potential for apoptosis imaging by means of PET and SPECT. However, thorough toxicologic studies are required before they can be applied in clinical studies. A further crucial factor is the timing of scanning after the start of treatment. In fact, the time course of apoptosis may differ depending on a large variety of factors, including apoptosis-inducing factor, tumor type, drug concentration, and exposure time. Consequently, in the clinical setting, it is crucial to initially characterize the time course of radiolabeled annexin A5 uptake so that the peak of apoptosis is detected and early tumor response to therapy can be evaluated.

To date, PS is the most promising biomarker for measuring the extent of cell death as an early readout of efficacy of anticancer therapy. Annexin A5 is the ligand with the highest affinity for PS. It has been used in molecular imaging protocols to measure cell death in vitro and in vivo in preclinical and clinical studies. Experiences with annexin A5 have indicated that aspects such as biodistribution and target to background ratio require further improvement. An approach is to stick to annexin A5 and improve biodistribution and the target to background ratio by changing annexin A5's primary structure and the label that is coupled to annexin A5. The pretargeting strategy also shows promise to achieve better target to background ratios. At the clinical level, various studies support the notion that ^99mTc-HYNIC-annexin A5 and SPECT allow for noninvasive, repetitive, quantitative apoptosis imaging and assessing tumor response as early as 24 hours after the start of the treatment. As PET offers higher resolution, higher sensitivity, and more accurate quantification when compared to SPECT, annexin A5 has also been labeled with ¹⁸F, ⁶⁴Cu, and ¹²⁴I. However, these labeling procedures proved laborious and time consuming, and the use of directly iodinated annexin A5 in a clinical setting has not proven to be suitable. More recently, attempts have been made to label annexin A5 with ⁶⁸Ga for PET as the short half-life of this radionuclide (68 minutes) minimizes the radiation dose to the patient and allows for rapid sequential imaging. As opposed to ⁶⁴Cu and ¹⁸F, ⁶⁸Ga can be eluted from an in-house Ga/Ge generator, and labeling procedures with this metal are straightforward and with specific chelating agents are feasible at room temperature. As ⁶⁸Ga behaves like ¹¹¹In, the great potential of labeling chelators is already available. Preclinical and clinical studies using these agents are awaited.

A novel promising SPECT apoptosis imaging tracer that has recently been described is ^99mTc-duramycin. Duramycin is the smallest known polypeptide of 19 amino acids and binds the membrane phospholipid phosphatidylethanolamine, which becomes externalized to the surface of apoptotic cells in a way similar to PS. The favorable in vivo imaging profiles of this new agent warrant further development and characterization. Another promising novel PET tracer for imaging of apoptosis is represented by ¹⁸F-ML-10 and has already proceeded to phase I/II clinical trials. However, the exact mechanism by which these probes target the apoptotic cell membrane is not known, and high doses are needed for imaging purposes. Consequently, further research is certainly recommended.

Although both BLI and NIRF have shown great potential in small-animal imaging, their usefulness for in vivo imaging in humans is limited to structures superficially located in the human body.

For the imaging of midterm to late apoptotic events such as DNA fragmentation and chromatin condensation, ultrasonography (US) has been performed. Recently, in 2008, the first preclinical tumor-based use of high-frequency ultrasound spectroscopy to noninvasively monitor tumor treatment in vivo took place. However, given that US cannot discriminate necrotic from apoptotic cells and is limited to superficially located tumors, it is questionable whether US will become a clinically useful tool in the assessment of therapy response.

The potential of MRS and MRI for imaging of late apoptotic processes is currently unclear. ¹H MRS signals from cytoplasmic lipids do not seem to be a unique identifier for apoptosis. Although ³¹P MRS can selectively detect and characterize metabolites in tumors (such as FBP and CDP-choline), a unique cancer signature on MRS has not (yet) been detected. Moreover, owing to a lack of standardization, the technique is not yet reproducible across research facilities. DW-MRI has been used as an alternative method to image apoptosis in response to chemotherapy. However MRI-measured ADC is also altered in response to therapies that induce nonapoptotic cell death, including necrosis and mitotic catastrophe, and an increase in ADC can occur owing to these two ways of cell death in the absence of apoptosis. Also, MRI suffers from a low sensitivity, requiring bulk injection of apoptosis-targeting agents.

Finally, two novel unclassified tracers, ApoSense and the related compound named NST-732, were shown to accumulate in apoptotic cells by an unknown mechanism. Although both agents have shown promising features, further investigation with both probes is warranted to further unravel their mechanism of action and applicability in human studies for predicting therapy response.

Conclusion

During the past decade, a lot of progress has been made in the field of apoptosis research, and novel apoptosistargeted anticancer drugs have been developed. However, the entire mechanism behind the apoptotic cascade is still unknown, and, undoubtedly, new apoptosis markers will soon be revealed. In the future, rapid progress in this important and exciting field of clinical research may be expected.

Footnotes

Acknowledgments

Financial disclosure of authors and reviewers: None reported.

References

Gerl

Vaux

. Apoptosis in the development and treatment of cancer. Carcinogenesis 2005;26:263–70, doi:10.1093/carcin/bgh283.

Reed

. Dysregulation of apoptosis in cancer. J Clin Oncol 1999;17:2941–53.

Hengartner

. The biochemistry of apoptosis. Nature 2000;407:770–6, doi:10.1038/35037710.

Leist

JÄÄttelä

. Four deaths and a funeral: from caspases to alternative mechanisms. Nat Rev Mol Cell Biol 2001;2:589–98, doi:10.1038/35085008.

Vermes

Haanen

Steffens-Nakken

Reutelingsperger

. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin V. J Immunol Methods 1995;184:39–51, doi:10.1016/0022-1759(95)00072-I.

Zwaal

Comfurius

Bevers

. Surface exposure of phosphatidylserine in pathological cells. Cell Moll Life Sci 2005;62:971–88, doi:10.1007/s00018-005-4527-3.

Corsten

Hofstra

Narula

. Counting heads in the war against cancer: defining the role of annexin A5 imaging in cancer treatment and surveillance. Cancer Res 2006;66:1255–60, doi:10.1158/0008-5472.CAN-05-3000.

Munoz

Frey

Pausch

. The role of annexin A5 in the modulation of the immune response against dying and dead cells Curr Med Chem 2007;14:271–7, doi:10.2174/092986707779941131.

Haberkorn

Kinscherf

Krammer

. Investigation of a potential scintigraphic marker of apoptosis: radioiodinated Z-Val-Ala-DL-Asp(O-methyl)-fluoromethyl ketone. Nucl Med Biol 2001;28:793–8, doi:10.1016/S0969-8051(01)00247-5.

10.

Faust

Wagner

Law

. The nonpeptidyl caspase binding radioligand (S)-1-(4-(2-[18]fluoroethoxy)-benzyl)-5-[1-(2-methoxymethylpyrrolidinyl) sulfonyl] isatin ([18F]CbR) as potential positron emission tomography-compatible apoptosis imaging agent. Q J Nucl Med Mol Imaging 2007;51:67–73.

11.

Scott

Adams

Chan

. Potent and selective nonpetide inhibitors of caspase 3 and 7 inhibit apoptosis and maintain cell functionality. J Biol Chem 2000;276:16007–14.

12.

Zhou

Chu

Rothfuss

. Synthesis, radiolabelling, and in vivo evaluation of an ¹⁸F-labelled isatin analog for imaging caspase-3 activation of apoptosis. Bioorg Med Chem Lett 2006;16:5041–6, doi:10.1016/j.bmcl.2006.07.045.

13.

Chen

Zhou

Chu

. Comparison of radiolabeled isatin analogs for imaging apoptosis with positron emission tomography. Nucl Med Biol 2009;36:651–8, doi:10.1016/j.nucmedbio.2009.03.008.

14.

Bauer

Bauder-Wuest

Mier

. ¹³¹I-labelled peptides as caspase substrates for apoptosis imaging. J Nucl Med 2005;46:1066–74.

15.

Laxman

Hall

Bhojani

. Noninvasive real-time imaging of apoptosis. PNAS 2002;99:16551–5, doi:10.1073/pnas.252644499.

16.

Coppola

Ross

Rehemtulla

. Noninvasive imaging of apoptosis and its application in cancer therapeutics. Clin Cancer Res 2008;14:2492–501, doi:10.1158/1078-0432.CCR-07-0782.

17.

Blankenberg

Katsikis

Tait

. In vivo detection and imaging of phosphatidylserine expression during programmed cell death. Proc Natl Acad Sci U S A 1998;95:6349–54, doi:10.1073/pnas.95.11.6349.

18.

Blankenberg

Katsikis

Tait

. Imaging of apoptosis (programmed cell death) with ^99mTc annexin V. J Nucl Med 1999;40:184–91.

19.

Ohtsuki

Akashi

Aoka

. Technetium-99m HYNIC-annexin A5: a potential radiopharmaceutical for the in-vivo detection of apoptosis. Eur J Nucl Med 1999;26:1251–8, doi:10.1007/s002590050580.

20.

Blankenberg

. To scan or not to scan, it is a question of timing: technetium-99m-annexin A5 radionuclide imaging assessement of treatment efficacy after one course of chemotherapy Clin Cancer Res 2002;8:2757–8.

21.

Takei

Kuge

Zhao

. Time course of apoptotic tumour response after a single dose of chemotherapy: comparison with ^99mTc-HYNIC-annexin A5 uptake and histological findings in an experimental model. J Nucl Med 2004;45:2083–7.

22.

Kemerink

Liu

Kieffer

. Safety, biodistribution, and dosimetry of ^99mTc-HYNIC-annexin A5, a novel human recombinant annexin A5 for human application. J Nucl Med 2003;44:947–52.

23.

Vermeersch

Loose

Lahorte

. ^99mTc-HYNIC-annexin A5 imaging of primary head and neck carcinoma. Nucl Med Commun 2004;25:259–63, doi:10.1097/00006231-200403000-00008.

24.

Van de Wiele

Lahorte

Vermeersch

. Quantitative tumour apoptosis imaging using technetium-99m-HYNIC annexin A5 single photon emission computed tomography. J Clin Oncol 2003;21:3483–7, doi:10.1200/JCO.2003.12.096.

25.

Vermeersch

Ham

Rottey

. Intraobserver, and day-today reproducibility of quantitative ^99mTc-HYNIC-annexin A5 imaging in head and neck carcinoma. Cancer Biother Radiopharm 2004;19:205–10, doi:10.1089/108497804323071986.

26.

Rottey

Van de Bossche

Siegers

. Influence of chemotherapy on the biodistribution of ^99mTc-hydrazinonicotinamide-annexin A5 in patients. Q J Nucl Med Mol Imaging 2009;53:127–32.

27.

Kartachova

Valdés Olmos

Haas

RLM

. ^99mTc-HYNIC-rh-annexin A5 scintigraphy: visual and quantitative evaluation of early treatment-induced apoptosis to predict treatment outcome. Nucl Med Commun 2008;29:39–44, doi:10.1097/MNM.0b013e3282f1bc22.

28.

Kartachova

van Zandwijk

Burgers

. Prognostic significance of ^99mTc-HYNIC-rh-annexin A5 scintigraphy during platinum-based chemotherapy in advanced lung cancer. J Clin Oncol 2007;25:2534–9, doi:10.1200/JCO.2006.10.1337.

29.

Kartachova

Haas

RLM

Valdés Olmos

. In vivo imaging of apoptosis by ^99mTc-annexin A5 scintigraphy visual analysis in relation to treatment response. Radiol Oncol 2004;72:333–9, doi:10.1016/j.radonc.2004.07.008.

30.

Rottey

Siegers

Van Belle

. Sequential ^99mTc-hydrazinonicotinamide-annexin A5 imaging for predicting response to chemotherapy. J Nucl Med 2006;47:1813–8.

31.

Guo

M-F

Zhao

Tian

. In vivo ^99mTc-HYNIC-annexin V imaging of early tumor apoptosis in mice after single dose irradiation. J Exp Clin Can Res 2009;28:136–44, doi:10.1186/1756-9966-28-136.

32.

Haas

RLM

de Jong

Valdés Olmos

. In vivo imaging of radiation-induced apoptosis in follicular lymphoma patients. Int J Radiat Oncol Biol Phys 2004;59:782–7.

33.

Hoebers

FJP

Kartachova

de Bois

. ^99mTc HYNIC-rh-annexin A5 scintigraphy for in vivo imaging of apoptosis in patients with head and neck cancer treated with chemoradiotherapy. Eur J Nucl Med Mol Imaging 2008;35:509–18, doi:10.1007/S00259-007-0624-X.

34.

Vanderheyden

Liu

. Evaluation of ^99mTc-MAG₃-annexin A5: influence of the chelate on in vitro and in vivo properties in mice. Nucl Med Biol 2006;33:135–44, doi:10.1016/j.nucmedbio.2005.09.002.

35.

Yang

Azdarinia

. In vivo and in vitro measurement of apoptosis in breast cancer cells using ^99mTc-EC-annexin A5. Cancer Biother Radiopharm 2001;16:73–83, doi:10.1089/108497801750096087.

36.

Kurihara

Yang

Cristofanilli

. Imaging and dosimetry of ^99mTc-EC-annexin A5: preliminary clinical study targeting apoptosis in breast tumours. Appl Radiat Isot 2008;66:1175–82, doi:10.1016/j.apradiso.2008.01.012.

37.

Kemerink

Boersma

Thimister

PWL

. Biodistribution and dosimetry of ^99mTc-BTAP-annexin A5 in humans. Eur J Nucl Med 2001;28:1373–8, doi:10.1007/s002590100578.

38.

Boersma

Liem

Kemerink

. Comparison between human pharmacokinetics and imaging properties of two conjugation methods for ^99mTc-annexin A5. Br J Radiol 2003;76:553–60, doi:10.1259/bjr/30385847.

39.

Belhocine

Steinmetz

Hustinx

. Increased uptake of the apoptosis-imaging agent ^99mTc recombinant human annexin A5 in human tumours after one course of chemotherapy as a predictor of tumour response and patient prognosis. Clin Cancer Res 2002;8:2766–74.

40.

Belhocine

Steinmetz

Green

. In vivo imaging of chemotherapy-induced apoptosis in human cancers. Ann N Y Acad Sci 2003;1010:525–9, doi:10.1196/annals.1299.097.

41.

Tait

Smith

Blankenberg

. Structural requirements for in vivo detection of cell death with ^99mTc-annexin A5. J Nucl Med 2005;46:807–15.

42.

Luo

Wang

Zhang

. Preparation and bioevaluation of ^99mTc-HYNIC-B1 as a novel radioligand for apoptosis imaging. Apoptosis 2008;13:600–8, doi:10.1007/s10495-008-0189-6.

43.

Fonge

de Saint Hubert

Vunckx

. Preliminary in vivo evaluation of a novel ^99mTc-labelled HYNIC-Cys-annexin A5 as an apoptosis imaging agent. Bioorg Med Lett 2008;18:3794–8, doi:10.1016/j.bmcl.2008.05.044.

44.

Greupink

Sio

Ederveen

Orsel

. Evaluation of a ^99mTc-labeled annexin A5 variant for non-invasive SPECT imaging of cell death in liver, spleen and prostate. Pharm Res 2009;26:2647–56, doi:10.1007/s11095-009-9981-4.

45.

Lahorte

CMW

Siegers

Philippé

. Synthesis and in vitro evaluation of ¹²³I-labelled human annexin A5. Biomed Eng 2001;17:51–3.

46.

Lahorte

CMW

Van de Wiele

Bacher

. Biodistribution and dosimetry study of I-123-rh-annexin A5 in mice and humans. Nucl Med Commun 2003;24:871–80, doi:10.1097/00006231-200308000-00005.

47.

Cornelissen

Lahorte

CMW

Kersemans

. In vivo apoptosis detection with radioiodinated annexin A5 in LoVo tumour-bearing mice following tipifarnib (Zarnestra, R115777) farnesyltransferase inhibitor therapy. Nucl Med Biol 2005;32:233–9, doi:10.1016/j.nucmedbio.2005.01.001.

48.

Zavitsanou

Nguyen

Greguric

. Detection of apoptotic cell death in the thymus of dexamethasone treated rats using [I123]annexin A5 and in situ oligonucleotide ligation. J Mol Hist 2007;38:313–9, doi:10.1007/sl0735-007-9104-7.

49.

Wen

Q-P

. Imaging taxane-induced tumour apoptosis using PEGylated, ¹¹¹In–labelled annexin A5. J Nucl Med 2004;45:108–15.

50.

Murakami

Takamatsu

Taki

. ¹⁸F-labelled annexin A5: a PET tracer for apoptosis imaging. Eur J Nucl Med Mol Imaging 2004;31:469–74, doi:10.1007/s00259-003-1378-8.

51.

Toretsky

Levenson

Weinberg

. Preparation of F-18 labelled annexin A5: a potential PET radiopharmaceutical for imaging cell death. Nucl Med Biol 2004;31:747–52, doi:10.1016/j.nucmedbio.2004.02.007.

52.

Zijlstra

Gunawan

Burchert

. Synthesis and evaluation of a ¹⁸F-labelled recombinant annexin A5 derivative, for identification and quantification of apoptotic cells with PET. Appl Radiat Isot 2003;58:201–7, doi:10.1016/S0969-8043(02)00302-0.

53.

Yagle

Eary

Tait

. Evaluation of ¹⁸F-annexin A5 as a PET imaging agent in an animal model of apoptosis. J Nucl Med 2005;46:658–66.

54.

Cauchon

Langlois

Rousseau

. PET imaging of apoptosis with ⁶⁴Cu-labelled streptavidin following pretargeting of phosphatidylserine with biotinylated annexin A5. Eur J Nucl Med Mol Imaging 2007;34:247–58, doi:10.1007/s00259-006-0199-y.

55.

Russell

O'Donoghue

Finn

. Iodination of annexin A5 for imaging apoptosis. J Nucl Med 2002;43:671–7.

56.

Glaser

Collingridge

Aboagye

. Iodine-124 labelled annexin A5 as a potential radiotracer to study apoptosis using positron emission tomography. Appl Radiat Isot 2003;58:55–62, doi:10.1016/S0969-8043(02)00239-7.

57.

Keen

Dekker

Disley

. Imaging apoptosis in vivo using ¹²⁴I annexin A5 and PET. Nucl Med Biol 2005;32:395–402, doi:10.1016/j.nucmedbio.2004.12.008.

58.

Collingridge

Glaser

Osman

. In vitro selectivity, in vivo distribution and tumour uptake of annexin A5 radiolabelled with positron emitting radioisotopes. Br J Cancer 2003;89:1327–33, doi:10.1038/sj.bjc.6601262.

59.

Dekker

Keen

Shaw

. Functional comparison of annexin A5 analogues labelled indirectly and directly with iodine-124. Nucl Med Biol 2005;32:403–13, doi:10.1016/j.nucmedbio. 2005.02.002.

60.

Dekker

Keen

Lyons

. MBP-annexin A5 radiolabelled directly with iodine-124 can be used to image apoptosis in vivo using PET. Nucl Med Biol 2005;32:241–52, doi:10.1016/j.nucmedbio.2004.11.006.

61.

Zhao

Beauregard

Loizou

. Non-invasive detection of apoptosis using magnetic resonance imaging and a targeted contrast agent. Nat Med 2001;7:1241–4, doi:10.1038/nm1101-1241.

62.

Schellenberger

Bogdanov

Jr Hogemann

. Annexin A5-CLIO: a nanoparticle for detecting apoptosis by MRI. Mol Imaging 2002;1:102–7, doi:10.1162/153535002320162769.

63.

Krishnan

Neves

de Backer

. Detection of cell death in tumours by using MR imaging and a gadolinium–based targeted contrast agent. Radiology 2008;246:854–62, doi:10.1148/radiol.2463070471.

64.

Neves

Krishnan

Kettunen

. A paramagnetic nanoprobe to detect tumour cell death using magnetic resonance imaging. Nano Lett 2007;7:1419–23, doi:10.1021/nl070126v.

65.

Petrovsky

Schellenberger

Josephson

. Near-infrared fluorescent imaging of tumour apoptosis. Cancer Res 2003;63:1936–42.

66.

Schellenberger

Bogdanov

Jr Petrovsky

. Optical imaging of apoptosis as a biomarker of tumour response to chemotherapy. Neoplasia 2003;5:187–92.

67.

Ntziachristos

Schellenberger

Ripoll

. Visualization of antitumour treatment by means of fluorescence molecular tomography with an annexin A5-Cy5.5 conjugate. PNAS 2004;101:12294–9, doi:10.1073/pnas.0401137101.

68.

Choi

Yessayan

Choi

. Quantitative analysis of chemotherapeutic effects in tumours using in vivo staining and correlative histology. Cell Oncol 2005;27:183–90.

69.

Edgington

Berger

Blum

. Noninvasive optical imaging of apoptosis by caspase-targeted activity-based probes. Nat Med 2009;15:967–73, doi:10.1038/nm.1938.

70.

Tunis

Czarnota

Giles

. Monitoring structural changes in cells with high-frequency ultrasound signal statistics. Ultras Med Biol 2005;8:1041–9, doi:10.1016/j.ultrasmedbio.2005.04.020.

71.

Czarnota

Kolios

Abraham

. Ultrasound imaging of apoptosis: high-resolution non-invasive monitoring of programmed cell death in vitro, in situ and in vivo. Br J Cancer 1999;81:520–7, doi:10.1038/sj.bjc.6690724.

72.

Banihashemi

Vlad

Debeljevic

. Ultrasound imaging of apoptosis in tumour response: novel preclinical monitoring of photodynamic therapy effects. Cancer Res 2008;68:8590–6, doi:10.1158/0008-5472.CAN-08-0006.

73.

Blankenberg

Katsikis

Storrs

. Quantitative analysis of apoptotic cell death using proton nuclear magnetic resonance spectroscopy. Blood 1997;89:3778–86.

74.

Lehtimaki

Valonen

Griffin

. Metabolite changes in BT4C rat gliomas undergoing ganciclovir-thymidine kinase gene therapy-induced programmed cell death as studied by 1H MRS in vivo, ex vivo and in vitro. J Biol Chem 2003;278:45915–23, doi:10.1074/jbc.M306209200.

75.

Schmitz

Kettunen

. 1H MRS-visible lipids accumulate during apoptosis of lymphoma cells in vitro and in vivo. Magn Reson Med 2005;54:43–50, doi:10.1002/mrm.20529.

76.

Valonen

Griffith

Vaisanen

. Macromolecular and lipid resonances in apoptosis BT4C glioma cells in vitro and tumours in vivo. Proc Int Soc Magn Reson Med 2002;10:26–34.

77.

Brindle

. Detection of apoptosis in tumours using magnetic resonance imaging and spectroscopy. Adv Enzyme Regul 2002;42:101–12, doi:10.1016/S0065-2571(01)00025-5.

78.

Brindle

. Molecular imaging using magnetic resonance: new tools for the development of tumour therapy. Br J Radiol 2003;76 Spec No 2:S111–7, doi:10.1259/bjr/50577981.

79.

Day

Kettunen

Gallagher

. Detecting tumour response to treatment using hyperpolarized 13C magnetic resonance imaging and spectroscopy. Nat Med 2007;13:1382–7, doi:10.1038/nm1650.

80.

Patterson

Padhani

Collins

. Technology insight: water diffusion MRI–a potential new biomarker of response to cancer therapy. Nat Clin Pract Oncol 2008;5:220–33, doi:10.1038/ncponc1073.

81.

Charles-Edwards

deSouza

. Diffusion-weighted magnetic resonance imaging and its application to cancer. Can Imaging 2006;13:135–43, doi:10.1102/1470-7330.2006.0021.

82.

Brauer

. In vivo monitoring of apoptosis. Prog Neuropsychopharmacol Biol Psychiatry 2003;27:323–31, doi:10.1016/S0278-5846(03)00026-5.

83.

Kim

Morgan

Zeng

. Diffusion-weighted MR imaging to assess early therapy with novel apoptosis-inducing anti-DR5 antibody. Radiology 2008;248:844–51, doi:10.1148/radiol.2483071740.

84.

Morse

Galons

Payne

. MRI-measured water mobility increases in response to chemotherapy via multiple cell-death mechanism. NMR Biomed 2007;20:602–14, doi:10.1002/nbm.1127.

85.

Kline

Petrylak

. Rapid in vivo monitoring of chemotherapeutic response using weighted sodium magnetic resonance imaging. Clin Cancer Res 2000;6:2146–56.

86.

Sharma

Katz

Taxotere chemosensitivity evaluation in mice prostate tumour: validation and diagnostic accuracy of quantitative measurement of tumour characteristics by MRI, PET, and histology of mice tumour. Technol Cancer Res Treat 2008;7:175–86.

87.

Sierra

Michaeli

Niskanen

J-P

. Water spin dynamics during apoptotic cell death on glioma gene therapy probed by T_1p and T_1p. Magn Reson Med 2008;59:1311–9, doi:10.1002/mrm.21600.

88.

Aloya

Shirvan

Grimberg

. Molecular imaging of cell death in vivo by a novel small molecule probe. Apoptosis 2006;11:2089–101, doi:10.1007/s10495-006-0282-7.

89.

Grimberg

Levin

Shirvan

. Monitoring of tumor response to chemotherapy in vivo by a novel small-molecule detector of apoptosis. Apoptosis 2009;14:257–67, doi:10.1007/s10495-008-0293-7.

90.

Reshef

Shirvan

Waterhouse

. Molecular imaging of neurovascular cell death in experimental cerebral stroke by PET. J Nucl Med 2008;40:1520–8, doi:10.2967/jnumed.107.043919.

91.

Shirvan

Reshaf

Allen

. Apoptosis imaging with PET-18F-ML-10 for early assessment of response of brain metastase to radiotherapy. J Nucl Med 2009;50 Suppl:453.

92.

Zeng

Yao

M-L

Townsend

. Synthesis, biological evaluation and radiochemical labelling of a dansylhydrazone derivates as a potential imaging agent for apoptosis. Biorg Med Chem Lett 2008;18:3573–7, doi:10.1016/j.bmcl.2008.05.002.

93.

Laumonier

Segers

Laurent

. A new peptide vector for molecular imaging of apoptosis identified by phage display technology. J Biomol Screen 2006;11:537–45, doi:10.1177/1087057106288220.

94.

Thapa

Kim

. Discovery of a phosphatidylserine recognizing peptide and its utility in molecular imaging of tumour apoptosis. J Cell Mol Med 2008;12:1649–60, doi:10.1111/j.1582-4934.2008.00305.x.

In Vivo Imaging of Apoptosis in Oncology: An Update

Abstract

Imaging Early Apoptotic Events: Caspase Activation

Radiolabelled Caspase Substrates/Inhibitors

Bioluminescence Imaging

Imaging Phospholipid Reorganization: PS Externalization

Annexin A5 SPECT Radionuclides

99mTc-HYNIC-Annexin A5

99mTc-MAG3-Annexin A5

99mTc-EC-Annexin A5

99mTc-BTAP-Annexin A5

99mTc-Annexin A5 Mutants

123I-Annexin A5

111In DTPA-PEG-Annexin A5

Annexin A5 PET Radionuclides

18F-Annexin A5

64Cu-DOTA-Biotin-Streptavidin Annexin A5

124I-Annexin A5

Magnetic Resonance Imaging

NIRF Imaging

Imaging Midterm to Late Apoptotic Events: DNA Fragmentation and Chromatin Condensation

Imaging Midterm to Late Apoptotic Events: Membrane Phospholipid Metabolism

Magnetic Resonance Spectroscopy

1H Magnetic Resonance Spectroscopy

31P Magnetic Resonance Spectroscopy

13C Magnetic Resonance Spectroscopy

Imaging Late Apoptotic Events: Cell Shrinkage and Membrane Blebbing

Magnetic Resonance Imaging

1H Magnetic Resonance Imaging

23Na Magnetic Resonance Imaging

Relaxation (T1/T2) MRI

Novel Apoptosis Tracers

Discussion

Conclusion

Footnotes

Acknowledgments

References