Molecular imaging of cell death in vivo by a novel small molecule probe

Abstract

Apoptosis has a role in many medical disorders, therefore assessment of apoptosis in vivo can be highly useful for diagnosis, follow-up and evaluation of treatment efficacy. ApoSense is a novel technology, comprising low molecular-weight probes, specifically designed for imaging of cell death in vivo. In the current study we present targeting and imaging of cell death both in vitro and in vivo, utilizing NST-732, a member of the ApoSense family, comprising a fluorophore and a fluorine atom, for both fluorescent and future positron emission tomography (PET) studies using an (18)F label, respectively. In vitro, NST-732 manifested selective and rapid accumulation within various cell types undergoing apoptosis. Its uptake was blocked by caspase inhibition, and occurred from the early stages of the apoptotic process, in parallel to binding of Annexin-V, caspase activation and alterations in mitochondrial membrane potential. In vivo, NST-732 manifested selective uptake into cells undergoing cell-death in several clinically-relevant models in rodents: (i) Cell-death induced in lymphoma by irradiation; (ii) Renal ischemia/reperfusion; (iii) Cerebral stroke. Uptake of NST-732 was well-correlated with histopathological assessment of cell-death. NST-732 therefore represents a novel class of small-molecule detectors of apoptosis, with potential useful applications in imaging of the cell death process both in vitro and in vivo.

Full text

Apoptosis (2006) 11:2089–2101
DOI 10.1007/s10495-006-0282-7
Molecular imaging of cell death in vivo by a novel small molecule
probe
Revital Aloya · Anat Shirvan · Hagit Grimberg ·
Ayelet Reshef · Galit Levin · Dvora Kidron ·
Avi Cohen · Ilan Ziv
Published online: 17 October 2006
C

Springer Science + Business Media, LLC 2006
Abstract Apoptosis has a role in many medical disorders,
therefore assessmentof apoptosisin vivocan behighly useful
for diagnosis, follow-up and evaluation of treatment efficacy.
ApoSense is a novel technology, comprising low molecular-
weight probes, specifically designed for imaging of cell death
in vivo. In the current study we present targeting and imag-
ing of cell death both in vitro and in vivo, utilizing NST-
732, a member of the ApoSense family, comprising a fluo-
rophore and a fluorine atom, for both fluorescent and future
positron emission tomography (PET) studies using an
18
F
label, respectively. In vitro, NST-732 manifested selective
and rapid accumulation within various cell types undergo-
ing apoptosis. Its uptake was blocked by caspase inhibition,
and occurred from the early stages of the apoptotic process,
in parallel to binding of Annexin-V, caspase activation and
alterations in mitochondrial membrane potential. In vivo,
NST-732 manifested selective uptake into cells undergoing
cell-death in several clinically-relevant models in rodents: (i)
Cell-death induced in lymphoma by irradiation; (ii) Renal
ischemia/reperfusion; (iii) Cerebral stroke. Uptake of NST-
Revital Aloya and Anat Shirvan are equal contribution to the paper
R. Aloya · A. Shirvan · H. Grimberg · A. Reshef ·G. Levin ·
A. Cohen · I. Ziv
NeuroSurvival Technologies (NST) Ltd.,
Petach-Tikva, Israel
A. Shirvan (

)
NeuroSurvival Technologies (NST) Ltd.,
5 Ha’Odem st, P.O. Box 7119,
Petach-Tikva 49170, Israel
e-mail: Anat@NST.co.il
D. Kidron
Department of Pathology, Meir Hospital,
Kfar-Saba, Israel
e-mail: dkidron@clalit.org.il
732 was well-correlated with histopathological assessment
of cell-death. NST-732 therefore represents a novel class of
small-molecule detectors of apoptosis, with potential useful
applications in imaging of the cell death process both in vitro
and in vivo.
Keywords Apoptosis
.
Cell death
.
Imaging
.
Chemotherapy
.
ApoSense
Introduction
Apoptosis is clearly one of the most fundamental biological
processes, and in recent years, cumulative data indicate that
apoptosis, its activation or dysregulation has a role in almost
any medical disorder, either in the etiology or pathogenesis
of disease [1–5]. Therefore, molecular imaging of apoptosis
can be a highly-useful tool in clinical medical practice. Such
imaging can assist in early diagnosis of disease [for exam-
ple in disorders associated with excessive activation of the
process (Alzheimer’s disease, Parkinson’s disease)], moni-
toring of disease course (e.g., myocardial infarction, cerebral
stroke), or monitoring of response to treatment, such as mon-
itoring of efficacy of anti-tumor agents for cancer, which op-
erate through induction of cell deathin thetumor tissue [6, 7].
It is therefore highly desirable to have tools for non-
invasive imaging of apoptosis in the clinical set-up. Prefer-
ably, such tool should be a low-molecular weight probe,
amenable for systemic administration in vivo, and capable of
performing selective detection of cells undergoing the death
process. Preferably, such probe would manifest uptake and
intracellular accumulation within the apoptotic cell, in or-
der to allow a high signal/noise ratio. The probe should also
be sensitive to detection of the death process from its early
stages. Currently, such tool is not available, which prompted
us to develop the ApoSense Technology.
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2090 Apoptosis (2006) 11:2089–2101
One of the earliest events in apoptosis is alterations in
the distribution of phospholipid constituents of the plasma
membrane [8, 9]. Healthy, viable cells manifest asymmetri-
cal distribution of phospholipids in the plasma membrane.
Phospholipid scrambling results in exposure of PS on the cell
surface, which can be detected by Annexin-V, a 36 kDa pro-
tein with high affinity to PS [10–12]. Annexin-V is a highly
useful probe in vitro, while its performance in vivo was found
to be limited [13], mainly due to pharmacokinetic problems
associated with it being a relatively-large protein, with very
slow clearance from the blood. In addition, Annexin-V in
vivo manifests a low signal/noise ratio, explained in part,
due to its binding only to the cell surface, while not mani-
festing uptake and intracellular accumulation within the cell
in the early stages of apoptosis.
ApoSense is a novel family of low-molecular weight am-
phipathic apoptosis markers,which target the cell membrane.
The compounds do not cross the plasma membrane of an in-
tact viable cell, but perform selective passage through the
membrane and accumulation within the cytoplasm of apop-
totic cells from the early stages of the death process. Uptake
of these compounds is parallel to the characteristic apoptotic
features of acquisition of Annexin-V binding, activation of
caspases and loss of mitochondrial membrane potential. Im-
portantly, uptake of the ApoSense compounds through the
cell membrane into the apoptotic cell precedes loss of mem-
brane integrity, assessed for example by propidium iodide
(PI) exclusion. We have previously reported on this perfor-
mance of one member of this family, DDC (N,N

-didansyl-
L-cystine, MW =707) in imaging of cell death in animal
models both in vitro and in vivo [14].
Positron emission tomography (PET) with a radio-label
such as
18
F is emerging as the leading modality for molec-
ular imaging, enabling non-invasive, sensitive, quantitative
and high-resolution imaging of biological processes. To meet
the challenge of PET imaging of apoptosis, we therefore
developed NST-732 [(5-dimethylamino)-1-napththalene-
sulfonyl-α-ethyl-fluoroalanine; (Fig. 1)], a member of the
ApoSense family, having a compact structure (MW =368)
and both a fluorophore (a dansyl group) and a fluorine atom
Fig. 1 Formula of NST-732
(potentially an
18
F isotope). While the fluorophore may al-
low fluorescent detection of binding of the compound to the
apoptotic cells,
18
F-labeled NST 732 may allow detection of
apoptosis in the clinical set-up, using PET. We now report
on the performance of NST-732, as assessed in fluorescence
studies, in detection of cell-death in vitro and in vivo,in
various cell lines and clinically-relevant animal models.
Materials and methods
Cell culture
Human adult T-cell Leukemia Jurkat cells (clone E6-1),
mouse lymphoma LY-S cells (L5178y-S) and CT26 colon
adenocarcinoma cells (CT26·WT) were obtained from
ATCC (Rockville, MD, USA). Cells were cultured in RPMI
1640 medium (Beit Haemek, Israel) supplemented with
2 mM of L-glutamine; 100 units/ml of penicillin; 100 µg/ml
of streptomycine; 12.5 units/ml nystatin; 1 mM sodium
pyruvate and 10% FCS. Both Jurkat and LY-S cells were
grown in suspension in vertical flasks and seeded at a density
of 5 ×10
6
cells in 10 ml medium. Mouse melanoma cells
(B16-F10, ATCC) were maintained in DMEM containing
high glucose (4.5 gr/l; Beit Haemek, Israel) supplemented
with 4 mM of L-glutamine; 100 units/ml of penicillin;
100 µg/ml of streptomycine; 12.5 units/ml nystatin and 10%
FCS. Cells were cultured in a humidified atmosphere con-
taining 5% CO
2
at 37
◦
C. Adherent CT26 and B16 cells were
grown in flasks and passaged by trypsinization every 2–3
days (seeded at a density of 1 ×10
6
cells in 10 ml medium).
Induction of apoptosis in vitro
Jurkat cells (1 ×10
6
cells/ml) were treated with IgM anti-Fas
antibody, CH11 (Medical and Biological laboratories, Japan)
at a concentration of 0.1 µg/ml for 120–180 min. Cells were
harvested and centrifuged at 1600 rpm for 10 min. CT26
and B16 cell lines were incubated for 16 h with 100 µM
BiCNU (Bristol-Myers Squibb, Syracuse, NY). Cells were
trypsinized, washed with PBS, centrifuged at 1000 for 3 min,
and taken for analysis.
Fluorescence microscopy
Cells (1 ×10
6
) were incubated with NST-732 (50 µMdis-
solved in NaPPi buffer, pH 7.4) or Annexin-V-FITC (IQ
Products, USA). Staining with Annexin-V was according to
the manufacture’s instructions for 20 min at RT in a volume
of 50 µl. PI (1:10) (IQ Products, USA) was added imme-
diately before microscopic analysis. Cells were visualized
using a fluorescent Olympus microscope (BX51TF; Olym-
pus Optical, U.K.), with UV illumination from a mercury
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Apoptosis (2006) 11:2089–2101 2091
lamp, using several fluorescence objectives. NST-732 and
PI staining were visualized with excitation at 365 nm and
emission at 420 (band pass) nm, while Annexin-V-FITC was
visualized with excitation at 488 nm and emission at 530 nm.
Fluorescent activated cell sorter (FACS) analysis
Apoptotic cells were detected by flow-cytometry after double
staining with either NST-732 and PI or Annexin-V-FITC and
PI. Briefly, at the indicated time points following induction of
apoptosis by the anti-Fas antibody, 30 µl of cell suspension
(approximately 1 ×10
6
/ml) were diluted in 300 µl HEPES
buffer (10 mM Hepes and 140 mM NaCl, pH 7.4) containing
Annexin-V-FITC/PI or 50 µM NST-732 and PI. After 20 min
of incubation at room temperature, cells were subjected to
analysis using FACS Vantage VE (BD Biosciences, San Jose,
CA) and CellQuest software (excitation for NST-732 was at
365 nm and emission was measured at 530 nm). A total of
10
4
events were collected for each sample.
Detection of caspase activation following apoptosis induced
by anti-Fas Ab
The methodology was based on the Fluorochrome Inhibitors
of Caspase (FLICA), using carboxyfluorescein-labeled flu-
oromethyl ketone peptide caspase inhibitor (FAM-VAD-
FMK), which produces a green fluorescence (CaspaTag
TM
pan-caspase in situ assay kit, Fluorescein, Chemicon,Temec-
ula, CA). FLICA binding specificity was tested by pre-
treatment with FAM-VAD-FMK according to the manufac-
turer instructions. Following one-hour incubation with FAM-
VAD-FMK, cells were washed, stained with PI and analyzed
by FACS.
Inhibition of apoptosis with caspase inhibitor
Jurkat cells were pre-incubated with PBS, in the presence
of 50 uM z-VAD-FMK in 0.05% DMSO (Enzyme system
products, Dublin, CA) or 0.05% DMSO for a control test,
followed by incubation with 0.1 ug/ml CH11 anti-Fas anti-
body. At 180 min, aliquots from each condition were stained
with NST732 and PI and analyzed by FACS.
Assessment of mitochondrial membrane potential by
TMRE
Assessment of mitochondrial trans-membrane potential was
performed using the lipophilic dye tetramethylrhodamine
ethyl ester-based (TMRE, Molecular probes, Oregon, USA)
[15–16] according to the manufacturer’s instructions. TMRE
dye displayed a red shift in excitation and emission fluores-
cence spectra upon delta psi (electric potential across the
inner mithochndrial membrane) driven mithochondrial up-
take [16]. At the indicated time periods following induction
of apoptosis by the anti-Fas Ab, 3 ×10
5
cells were incu-
bated with 100 nM TMRE at room temperature for 20 min
in HEPES buffer, and the cellular red TMRE fluorescence
was detected using FACS (Excitation was at 488 nm and
emission was measured at 580 nm).
Renal ischemia and reperfusion injury in rats
Spraque-Dawley rats, weighing 180–250 gr (Harlan labora-
tories, Jerusalem, Israel)were anesthetized by intraperitoneal
injection of the combination of Ketamine, 80 mg/kg and Xy-
lazine, 10 mg/kg. Renal iscemia was performed according
to Liebethal, et al. [17]. After a midline laparatomy incision,
the left renal artery was isolated and clamped for 45 min,
causing renal ischemia. Reperfusion was then allowed for
24 h. Animals were then injected intravenously with NST-
732 (35 mg/kg) for a time period of 2 h and both kidneys
were excised, frozen in liquid nitrogen stored at −80
◦
C and
further submitted for histological sections.
Experimental cerebral stroke due to middle cerebral artery
(MCA) occlusion in mice
Cerebral ischemiawas induced in Balb/C mice (10–12 weeks
old, Harlan, Jerusalem) by MCA cauterization. Mice were
anesthetized, and the temporal bone was exposed. Scraping
of the bone up to a minimal hole allowed exposure of the
MCA subjecting it to cauterization. After 22 h from the
insult, NST-732 (70 mg/kg) was injected intravenously, two
hours before sacrificing the animals. At 24 h from induction
of the injury, mice were sacrificed by anesthetic overdose,
and brains were removed into liquid nitrogen for further
histopathology.
Lymphoma model in mice
DBA mice (6–8 weeks old males, Jackson laboratories) were
injected subcutaneously with 10
6
LY-S cells in 50 µl saline,
and were examined daily for tumor formation. For irradi-
ation therapy, mice were irradiated on days 8, 9 and 10,
once daily, with 6MV X-rays in Linac apparatus in 3 frac-
tions of 6 Gray/day. Mice received a dose of 1.0 centi-Gray
per monitor unit. At day 13 (72 h following the last ir-
radiation) mice were injected intravenously with NST-732
(70 mg/kg) dissolved as above. Two hours later, tumors were
excised, frozen in liquid nitrogen and frozen sections were
prepared.
Preparation of histological sections and TUNEL analysis
Four µm thick sections were prepared for microscopic anal-
ysis, to follow cells manifesting uptake of NST-732, under
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2092 Apoptosis (2006) 11:2089–2101
UV fluorescence objective. Consecutive slides were stained
with Hematoxylin and Eosin (H&E) or with TUNEL (Termi-
nal Deoxynucleotidyltransferase-mediated dUTP Nick End
Labeling) [18]. The ApoTag in situ apoptosis detection flu-
orescent kit (Chemicon International, Temecula, CA) was
also used, according to manufacturer’s instructions.
Preparation of cytosolic extracts from tumors and
quantification of NST 732 binding
Excised tumors were weighted and homogenized (using
Heidolph RZR 2020 homogenizer, Heidolph instruments
GmbH & Co.KG, Schwabach, Germany) in cold homog-
enization buffer containing Tris HCL 50 mM and 0.005%
Triton, pH =7.6 (1:7 w/v). Following total homogeniza-
tion, samples were centrifuged twice at 13,800 rpm for
20 min at 4
◦
C and the supernatants were collected and
stored at −80
◦
C. For quantification of NST-732 uptake,
homogenized tumor samples were aliquoted (in triplicates)
onto black Ritter flat bottom micro plates, and read at
360 nm excitation and 520 nm emission, using fluorescence
micro plate reader (GENious Fl Reader, Tecan, Grodig,
Austria). A linear calibration curve was performed using
NST-732 at concentrations between 0–25 µg/ml. Based on
the calibration curve accumulation of NST-732 (expressed
as µg/g tumor tissue) was calculated.
Toxicological studies
Mice were subjected to an administration of NST-732 in a
single dose, up to a dose of 275 mg/kg. In addition, an acute
extended single-dose toxicological study was performed in
mice (n =5) at the dose of 5 mg/kg, a dose being more
that 2500-fold higher than the expected human dose for
18
F-
radiolabled NST-732 for clinical PET studies. Mice were
evaluated at 24 h, 3 days and 14 days post dosing for blood
hematology and chemistry, and histopathology assessment
was performed on day 14.
Statistical analysis
Student’s t-test was used to assess quantitative differences in
uptake of NST-732 between control and treated tumors. Sta-
tistical significance was defined as p<0.05. For qualitative
microscopic and flow-cytometric observations, representa-
tive results out of at least 3 independent experiments are
presented.
Results
NST732 detects apoptosis in various cell types and in
response to various apoptotic triggers
Figure 2 shows fluorescent microscopy of cultured B16
melanoma cells, before and after exposure to BiCNU
(100 µM, 16 h). As shown, the control culture contained
very few cells manifesting uptake of NST-732 (green flu-
orescence), while most of the cells manifested only back-
ground cellular blue auto-fluorescence, usually observed in
response to UV illumination (Fig. 2A). The cells manifest-
ing uptake of NST-732 reflect cell death, normally occurring
in cell cultures. These cells were also stained with PI, thus
representing cells in the late stages of cell death, wherein
Fig. 2 Detection of Cell death by NST-732 in B16 melanoma cells
in vitro; fluorescent microscopy, co-staining with propidium iodide
(PI). A. Control cells; most cells are unstained, manifesting only blue
auto-fluorescence (blue arrow), un-related to NST-732; few cells man-
ifest binding of both NST-732 and PI (red arrow), reflecting naturally-
occurring cell death in culture. B. Following incubation with BiCNU
(100 µM, 16 h), numerous cells manifest uptake of NST-732 (green flu-
orescence). Some of the cells show uptake of NST-732, while excluding
PI. These are cells in early apoptosis (EAC; green arrow). Some of the
cells show uptake of both NST-732 and PI (red arrow), being cells in
the late stages of apoptosis or undergoing a necrotic mode of cell death.
Scale bar: 1 cm =80 µm
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Apoptosis (2006) 11:2089–2101 2093
Fig. 3 FACS analysis of uptake of NST-732 by Jurkat cells under-
going apoptosis induced by anti-Fas Ab. A. Control cells (solid line)
versus apoptotic cells (dashed line). Analysis of 10
4
cells after incuba-
tion with NST-732 (50 µM). Apoptosis was associated with a shift of
the cell population to a new and distinct peak of higher fluorescence,
reflecting enhanced uptake of NST-732 upon induction of apoptosis. B,
C. Co-staining of NST-732 versus propidium iodide (PI); control cells
(B) versus apoptotic cells (C). As shown, most of the cells undergoing
apoptosis induced by anti-Fas Ab were in the early stages of the process
(EAC), manifesting enhanced uptake of NST-732, while excluding PI
(right lower quadrant). This shows the capability of NST-732 to per-
form selective uptake into apoptotic cells at an early stage, wherein
membrane integrity is still maintained. In addition, NST-732 also de-
tected the cells in the late stages of the apoptotic process, manifesting
uptake of both NST-732 and PI. The figure describes a representative
experiment out of ten performed
the membrane loses its integrity, thus enabling ingress of PI.
By contrast, upon exposure to the chemotherapeutic drug
BiCNU (Fig 2B), numerous cells took up NST-732. Im-
portantly, the compound accumulated within the cytoplasm.
Staining of apoptotic cells by NST-732 was stable, and was
not reduced by additional washing, suggesting that the up-
take is irreversible. Also important is the fact that many of
the cells constituting this new population of NST-732-stained
cells did not stain with PI. These cells are hereinafter des-
ignated Early-Apoptotic Cells (EAC). NST-732 is therefore
capable of detecting cells in the early stage of apoptosis,
before loss of membrane integrity.
The phenomenon of EAC was constantly observed also
with various other cell types and triggers of apoptosis, for
example HeLa cells (cervical tumor-derived cells) treated
with staurosporine (data not shown), CT26 colon carcinoma-
derived cells, treated with BiCNU (Fig. 4B), and Jurkat cells
induced to undergo apoptosis by treatment with anti-Fas an-
tibody (Fig. 3). Detection of EAC by NST 732 was further
characterized by FACS. Figure 3A shows that treatment with
anti-Fas antibody created a shift of the cell population, with
formation of a new and distinct peak, characterized by higher
fluorescence levels. Dot-plot analysis revealed that the new
cell population consists mainly of PI-excluding cells, i.e.,
cells in early apoptosis (Fig. 3B).
Uptake of NST732 binding is correlated to the activation of
the membrane flip-flop mechanism
Sensitivity of the uptake of NST-732 to activation of
the flip-flop of membrane constituents occurring in early
apoptosis was tested by focusing on EAC, and comparing
the time-course of NST-732 uptake by these cells, to the
binding of fluorescent-labeled Annexin-V. Annexin-V has
high affinity to the headgroups of phosphatidylserine (PS)
[10–12]. Since exposure of PS on the cell surface occurs
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2094 Apoptosis (2006) 11:2089–2101
Fig. 4 Detection of early apoptotic events by NST-732: correlation
with Annexin-V. A. Time course of binding of NST-732 and Annexin-V
to Jurkat cells induced to undergo apoptosis by treatment with anti-Fas
Ab. Flow cytometry was performed at different time points after apop-
totic induction with anti-Fas Ab. Staining was performed with either
NST-732 or annexin-V, versus PI. EAC were identified as cells man-
ifesting NST-732 or Annexin-V uptake while excluding PI. 10
4
cells
were counted at each time point. As shown, time-course curves for
detection of apoptosis by NST-732 and Annexin-V were practically
identical. B. CT26 murine colon carcinoma cell, undergoing apopto-
sis induced by BiCNU; fluorescent microscopy showing co-staining
with NST-732 (green) and Annexin-V Cy3 (red). While Annexin-V
localized to the cell membrane, NST-732 manifested accumulation in
the cytoplasm. It is noteworthy, that NST-732 was excluded from the
nucleus. Scale bar: 1 cm =6 µm
only upon activation of the membrane flip-flop mechanism,
binding of exogenously-administered Annexin-V can serve
as a reliable reporter on said membrane flip-flop. In the repre-
sentative experiment described in Fig. 4A, Jurkat cells were
induced to undergo apoptosis by treatment with anti-Fas Ab.
EAC were detected by flow cytometry as PI-excluding cells.
The temporal profile of binding of Annexin-V or uptake
of NST-732 by these EAC after initiation of the apoptotic
trigger was co-measured thereafter. As shown, the temporal
profile was practically identical. Thus, uptake of NST-732
in early apoptosis, and the apoptotic plasma membrane
flip-flop are closely-related. Fig. 4B further demonstrates
this observation, showing fluorescent microscopy of a C26
colon carcinoma cell, in the early stages of apoptosis induced
by the anticancer agent BiCNU. The cell was co-stained
with both fluorophore-labeled Annexin-V (red fluorescence)
and NST-732 (green fluorescence). Characteristically,
Annexin-V binds only to the surface of the EAC membrane,
where it binds to the exposed PS headgroups. Therefore, it
shows a ring-like peripheral staining. By contrast, in parallel
to the binding of Annexin-V to the cell surface, NST-732
accumulates within the cytoplasm of the early apoptotic
cell.
Uptake of NST-732 is related to caspase activation
Previous studies have shown that interaction through the
death receptor CD95 induces apoptosis by formation of a
signaling complex at the cell membrane and subsequent
caspase-8 and caspase-3-activation [19]. Therefore, the sen-
sitivity of uptake of NST-732 to the apoptotic process was
tested in Jurkat cells, induced to undergo apoptosis by anti-
Fas antibody, but concomitantly treated with the broad-
spectrum caspase inhibitor Z-VAD-FMK. As shown in a
representative experiment in Fig. 5A, addition of the cas-
pase inhibitor resulted in a marked shift of the fluorescent
intensity to lower levels, leading to a peak very similar to
that of the control cells, suggesting maximal inhibition (Fig.
5A). Thus inhibition of the apoptotic process by a caspase
inhibitor blocked uptake of NST-732 by these cells, in spite
of their exposure to the pro-apoptotic trigger. Further char-
acterization of these cells by co-staining with PI, revealed
that this dramatic effect of caspase inhibition in blocking
uptake of NST-732 was indeed on the EAC: In the control
culture, the percentage of EAC was 9%, and upon treatment
with the anti-Fas Ab, it rose dramatically to 75%. However,
this uptake by the EAC was totally blocked by concomitant
treatment with Z-VAD-FMK, to the level of 7.5% (Fig. 5B).
To further study the relationship between NST-732 uptake
by the apoptotic cells and caspase activation, we compared
the time-course of uptake of NST-732 into Jurkat cells from
initiation of exposure to the apoptotic trigger anti-Fas-Ab,
with the respective time-course of uptake of the fluorogenic
caspase substrate FAM-VAD-FMK. As shown in Fig. 6A,
acquisition of NST-732 uptake by the apoptotic cell was par-
allel to caspase activation, and at 120 min exposure to the
apoptotic trigger, both NST-732 and the caspase substrate
recognized an equal percentage of cells, i.e., at that time
point, update of NST-732 matched caspase activation. In-
terestingly however, at earlier stages of apoptosis induction,
NST binding seems to precede detection by the fluorogenic
substrate: for example, at sixty minutes, 30% of the cells al-
ready bound NST-732, while only few events were detected
by the caspase substrate. This finding suggests that uptake of
NST-732 precedes activation of the caspases detectable by
FAM-VAD-FMK.
Uptake of NST-732 is correlated with mitochondrial
membrane potential disruption
One of the major events of the apoptotic cascade is disruption
of the mitochondrial membrane potential. In order to study
the correlation between uptake of NST-732 and changes in
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Apoptosis (2006) 11:2089–2101 2095
Fig. 5 Inhibition of NST-732 binding by the caspase inhibitor z-VAD-
FMK. Jurkat cells were treated with anti-Fas antibody in the presence
or absence of the caspase inhibitor z-VAD (50 µM). Following 150 min
of incubation, cells were washed and stained with NST-732 (50 µM).
PI was added for detection of cells in early apoptosis, and cells were
subjected to flow cytometry. A. Uptake of NST-732 by control cells
(densely-dotted line) versus cells induced to undergo apoptosis by anti-
Fas Ab, (dotted line), and cells treated with the apoptosis inducer, but in
the presence of the caspase inhibitor (solid line). B. Quantitative analy-
sis of the percentage of cells in early apoptosis (i.e., NST-732-positive,
PI-negative cells). As shown, apoptosis was associated with a marked
shift of the cell population to a distinct peak of higher fluorescence, re-
flecting uptake of NST-732 by the apoptotic cells. However, inhibition
of the apoptotic process by the caspase inhibitor, practically entirely
blocked the uptake of NST-732
the mitochondrial membrane potential, Jurkat cells were in-
duced to undergo apoptosis by treatment with Anti-Fas Ab,
and the temporal profile of uptake of NST-732 was com-
pared with the signal obtained from tetramethylrhodamine
ethyl ester-based (TMRE), a potentiometric fluorescent dye
that incorporates into the mitochondria and thus able to mea-
sure and report on the mitochondrial membrane potential in a
semi-quantitative manner [15, 16]. Exposure to the Anti-Fas
Ab apoptotic trigger led to a progressive decline in mito-
chondrial membrane potential as reported by TMRE, with
a half-life of 75 min. Uptake of NST-732 mirrored this de-
cline in mitochondrial membrane potential (Fig. 6B), and at
the time point of 140 min, all cells in culture that showed
NST-732 uptake also showed loss of their mithochondrial
membrane potential. Interestingly however, acquisition of
NST-732 uptake by the cells seemed to precede mitochon-
drial potential breakdown to some extent: for example, sixty
minutes post-treatment with anti-Fas antibody, most of the
cells (91%) still retained TMRE and only later indicated dis-
ruption of mitochondrial membrane potential, while at that
time, a large portion of the cells (58.45%) already indicated
their apoptotic destiny by binding NST-732.
NST-732 is a detector of cell death in vivo in various animal
models of apoptosis
In order to exemplify the potential utility of NST-732 in
detection of cell death in pathological cases, clinically-
relevant animal models were chosen, wherein apoptosis
plays a role in the pathogenesis of disease: renal is-
chemia/reperfusion, and cerebral stroke. In addition, a cancer
model, in which detection of cell death may be beneficial for
the assessment of tumor response to treatment, was cho-
sen: lymphoma-bearing mice, treated with irradiation. In all
these models, NST-732 was administered intravenously, an-
imals were sacrificed thereafter, and the selective uptake of
NST-732 only by cells undergoing cell death was demon-
strated at the single-cell or whole-organ level, by fluorescent
detection.
Cell death of tubular cells following renal
ischemia-reperfusion (IR) injury
Unilateral renal ischemia/ reperfusion injury was induced
in rats by transient (45 min) ligation of the renal artery.
After reperfusion, NST-732 was administered intravenously
as described above. Marked accumulation of NST-732 in
the damaged ischemic kidney, but not in the control in-
tact kidney was observed. This was evident at the level
of whole-organ imaging (Fig. 7A), showing multiple foci
of increased uptake of NST-732, present only in the is-
chemic kidney, but not in the contralateral kidney. Histolog-
ical studies by fluorescent microscopy following H&E and
TUNEL staining confirmed that these foci of increased NST-
732 uptake were indeed foci of cells undergoing cell death
(Fig. 7 B–D).
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2096 Apoptosis (2006) 11:2089–2101
Fig. 6 Correlation of NST-732 uptake with key events in the apoptotic
cascade: caspase activation and mitochondrial membrane potential dis-
ruption. Jurkat cells were induced to undergo apoptosis by treatment
with anti-Fas antibody, and the time-course of NST-732 uptake by the
cells was monitored and correlated with key events in the process: A.
caspase activation, detected by the caspase substrate FAM-VAD-FMK;
B. disruption of mitochondrial membrane potential, detected by the
potential-sensitive probe TMRE (see text for details). As shown, NST-
732 uptake into the apoptotic cells was closely correlated with, and even
tended to precede, caspase activation and mitochondrial membrane al-
terations
Detection of cerebral neuronal cells undergoing cell death
MCA in mice
Cerebral ischemia was induced in mice by unilateral (left)
permanent occlusion by cauterization of the MCA. At 24 h
after insult, mice were subjected to an intravenous injec-
tion of NST-732. Accumulation of NST-732 in the ischemic
area was visualized at the level of whole-organ imaging by
stereomicroscope, and is shown in Fig. 8A. Highly selective
uptake of NST-732 in the left hemisphere was detected, only
in the region supplied by the MCA, while no such uptake
was observed in the contralateral hemisphere. Interestingly,
the core of the infarct manifested markedly increased up-
take, exemplified by a brighter staining (yellow arrow), as
compared to the more peripheral regions (red arrowheads).
In a microscopic analysis, the region of the infarct showed a
high number of individual cells manifesting uptake and in-
tracellular accumulation of NST-732, creating a “starry-sky
appearance” (Fig. 8B and C), in clear contrast to the adja-
cent intact regions, not affected by the ischemic insult, which
did not manifest such uptake. Co-staining with mouse anti-
neuronal nuclei (NeuN) monoclonal antibody confirmed the
neuronal identity of the cells (data not shown). TUNEL stain-
ing confirmed that the cells manifesting uptake by NST-732
were indeed cells undergoing cell death, showing the charac-
teristic apoptotic DNA fragmentation detectable by TUNEL
(Fig. 8D).
Detection by NST-732 of therapy-induced tumor cell death
in tumor-bearing mice
Lymphoma-bearing mice were subjected to radiotherapy
as described above. NST-732 was then administered intra-
venously, and two hours later, animals were sacrificed. Tu-
mors from control non-irradiated animals and irradiated ani-
mals were subjected to whole-organ fluorescent imaging. As
showninFig.9A, the non-irradiated tumor contained only
several small foci of NST-732 uptake tumor. However, upon
induction of cell death via irradiation, a dramatic increase in
NST-732 uptake occurred. Fluorescent microscopy (Fig. 9B)
of an area of cells showing high NST-732 uptake, in compar-
ison with Hematoxilin/Eosin (H/E) staining performed on a
consecutive slide (Fig. 9C), indicated that this was indeed
an area with disrupted tissue integrity containing numerous
cells undergoing cell death, characterized by cytoplasmic
acidification and chromatin condensation. TUNEL staining
(Fig. 9D) further showed that uptake of NST-732 and posi-
tive TUNEL staining were co-localized, confirming that the
cells labeled with NST-732 were indeed cells undergoing a
death process.
The uptake of NST-732 by the irradiated vs. control tu-
mors was quantified as described above. As shown in Fig. 10,
irradiation caused a dramatic increase in uptake of NST-732.
While low uptake of NST-732 was observed in the control
group [0.65 ± 0.2 µg/gr tumor (mean ± SEM)], irradiated
tumors manifested a 12-fold increase in NST-732 uptake, to
a level of 7.93 ± 1.58 µg/gr tumor (P<0.001).
Toxiclogical studies
Administration of NST-732, in a single dose, up to a dose of
275 mg/kg was not associated with any observable adverse
effects. The detailed acute single dose toxicological study,
performed at a dose of 5 mg/kg (more than 2500-fold higher
than the expected human dose for
18
F-radiolabled NST-732
for PET) did not show any significant clinical signs in the
blood tests (hematology and chemistry) over a 14-day obser-
vation period. The mice had a normal gain in body weight
compared to control group, and no abnormality was found
in their postmortem histopathological examination. These
results indicate a good safety profile for NST-732.
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Apoptosis (2006) 11:2089–2101 2097
Fig. 7 Fluorescent imaging of apoptosis by systemic administration
of NST-732 in vivo following renal ischemia/reperfusion injury. Rats
were subjected to unilateral clamping of the renal artery for 45 min,
followed by 24 h of reperfusion. A. Ex-vivo fluorescent whole-organ
imaging of the ischemic and the control kidneys. B–D. Histological
findings in the ischemic kidney: B & C: accumulation of NST-732 in
single tubular cells, (D) verification by TUNEL that cells stained by
NST-732 are undergoing cell death. Scale bars: (A)1 cm =125 µm; (B)
1cm=25 µm; (C) 1 cm =50 µm
Discussion
The emerging role of apoptosis in almost any medical dis-
order, either in the etiology or pathogenesis of disease, calls
for development of tools for detection of cell death in vivo,
in the clinical set-up. While numerous probes for cell death
are available in vitro, targeting various stages of the apop-
totic process, a barrier currently exists in their use in vivo.
Among others, the in vivo situation adds the dimensions of
toxicity, immunogenicity, and pharmacokinetics, all leading
to the exclusion of practically any probe for apoptosis de-
veloped to date. The ApoSense approach was developed to
overcome this barrier, by a rationale combining the following
considerations: (i) Aiming at a cellular target, which is well-
accepted as an early event of apoptosis. Preferably, this target
should be on the cell surface, thus providing easy accessibil-
ity of a probe administered systemically in vivo; (ii) Probing
this target by a low-molecular weight agent. Such agents, in
contrast to large proteins like antibodies or Annexin-V, are
characterized by relatively more favorable bio-distribution
and clearance, and are more amenable for structural opti-
mization, all important features in construction of a useful
imaging agent; (iii) A probe that will perform intracellular
accumulation, thus providing high signal/noise ratio; and (iv)
A non-toxic probe suitable for systemic administration.
NST-732 was built in accordance with the above
guidelines. It is a novel low molecular weight compound
(MW =368 Da), which manifests selectivity in binding to
apoptotic cells, remarkably identical to that of the relatively
large protein Annexin-V. As shown in this paper, detection
of cell death by NST-732 was found to be universal, i.e.,
irrespective of cell type or apoptotic trigger, and was found
to occur early in the apoptotic process, before disruption
of the continuity of the plasma membrane. Uptake of
NST-732 is parallel (or perhaps even precedes) caspase
activation, and can be completely blocked when the pro-
apoptotic trigger is co-administered with a caspase inhibitor.
Time-course of NST-732 uptake is also parallel (or even
slightly precedes) the characteristic apoptotic mitochondrial
membrane potential disruption. Taken together, NST-732
is an apoptosis-sensitive probe, which uptake accurately
corresponds with the induction of the death process or its
inhibition, and which also accurately reflects molecular
events associated with the apoptotic process.
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2098 Apoptosis (2006) 11:2089–2101
Fig. 8 Fluorescent imaging of apoptosis in acute cerebral stroke by
systemic administration of NST-732 in vivo. MCA cauterization was
performed in mice as detailed under materials and methods. NST-732
was administered intravenously, and two hours later, animals were sac-
rificed, and brains were sectioned, subjected to whole-organ imaging
and histological assessment. A. Whole-organ imaging, showing intense
uptake of NST-732 in the hemispheric region, corresponding with the
MCA distribution. Core of the infarct is characterized by more intense
signal (marked by yellow arrow), and peripheral damaged areas shows
lower intensity of staining (marked by red arrowheads). By contrast,
no signal was obtained for other brain regions. (B) Histological analy-
sis revealed that the signal originated from numerous individual cells,
manifesting intense intracellular accumulation of NST-732. Shown is
an area nourished by the MCA, containing a high density of cell death,
taken from the small red frame marked in (A). Note the clear border
formed between the intact live tissue and the damaged tissue. (C). An
area with a low density of cell death, representing the margins of the
injured tissue. (D). TUNEL ex-vivo staining of a consecutive slide as in
C, confirmed the identity of these cells as cells undergoing cell death.
Scale bars: (A)1 cm =125 µm; (B) 1 cm =50 µm
Both Annexin-V and NST-732 are capable of detecting
cells in early apoptosis via membrane associated processes:
Annexin-V by binding to the PS headgroups, and NST-732
by crossing the intact membrane into the cell. Both mech-
anisms of detection are selective for the death process, as
no binding of Annexin-V or uptake of NST-732 are ob-
served in viable cells. However, such mechanism indicates
that stages or modes of cell death wherein membrane dis-
ruption occurs, such as cells in the late stages of apoptosis,
or cells undergoing a necrotic mode of cell death, also man-
ifest uptake of either annexin-V or NST-732. In summary,
both Annexin-V and NST-732 are highly specific for cells
undergoing cell death, and are excluded from viable cells.
Both probes have the unique capability to detect cells in the
early stages of apoptosis, in which membrane integrity is
maintained, and their binding is parallel to other apoptotic
molecular events (e.g, caspase activation, and disruption of
mitochondrial membrane potential). Both probes follow the
entire apoptotic process, covering its entire scope. Never-
theless, modes or stages of cell death wherein membrane
disruption occurs will also be detected by either NST-732 or
Annexin-V.
The exact mechanism by which NST-732 targets the cell
membrane of the apoptotic cells and crosses it in early apop-
tosis should be further elucidated. A plausible mechanism
which merits further exploration, is a potential association
with the dramatic process of scrambling of membrane phos-
pholipids, that occursearly in apoptosis. ApoSensemolecules
all share amphipathic structures, having specific hydropho-
bic and charged moieties (see Fig. 1 for NST-732). Extensive
structure/function analyses that we performed (to be pub-
lished separately) have confirmed that integration of all these
moieties is required to enable performance of these com-
pounds as detectors of apoptosis. The hydrophobic moiety
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Apoptosis (2006) 11:2089–2101 2099
Fig. 9 Uptake of NST-732 in vivo into B16 melanoma cells under-
going cell death. Lymphoma (LY-S) tumors were established in DBA
mice. Mice were then treated by irradiation as described in the text.
NST-732 was administered intravenously 72 h after the last dose of
irradiation. Two hours later, mice were sacrificed, and tumors were sub-
jected to whole-tumor and microscopic analysis. A. Ex-vivo imaging of
the lymphoma control (non-radiated) tumor as compared to irradiated
tumor, showing dramatic increase of NST-732 uptake upon irradiation.
B. Fluorescent microscopy of an intense area of cell-death within the
irradiated tumor, showing that the signal originated from intracellular
accumulation of NST-732 in multiple individual cells undergoing cell
death. C.H/Eex-vivo staining of a consecutive slide, showing that
the uptake of NST-732 is in high correlation with area of cell death. D.
TUNEL ex-vivo staining of a consecutive slide, showing co-localization
of the cell-death TUNEL staining with NST-732 uptake, Scale bars: (A)
1cm=300 µm; (B,C,D) 1 cm =100 µm
seems to provide a membrane anchor, while the charged moi-
ety acts to prohibit crossing the highly hydrophobic mem-
brane core in viable cells. The scrambling process, initiated
early in apoptosis and mediated, at least in part, by the scram-
blase protein(s), substantially reduces this energetic barrier
[20]. Consequently, and similar to the respective flip-flop of
native phospholipid molecules, the scrambling process may
potentially allow flip-flop of NST-732 from the outer to the
inner membrane leaflet, and thus its ingress into the cell.
NST-732 manifests several potential advantages over the
current state-of-the-art in the field. The main advantage is the
intracellular accumulation of the compound, from the early
stages of the apoptotic process. This may entail a stronger
signal, as compared to Annexin-V, which binds only periph-
erally, to the external leaflet of the cell membrane. Indeed,
a thermodynamic study performed in vitro, wherein Jurkat
cells were induced to undergo apoptosis by anti-Fas antibody,
revealed that the number of NST molecules accumulated per
cell is 1.5 ×10
8
(not shown). This is about two orders of
magnitude higher than the calculated capacity of an apop-
totic cell for Annexin-V binding [about 4 ×10
6
molecules
per cell [21, 22]]. This high number of NST-732 molecules
accumulating per apoptotic cell may beneficially contribute
to enhancement and amplification of the signal obtained in
imaging studies. Another advantage of the compound is that
while Annexin-V manifests non-specific binding to the renal
cortex, NST-732 does not have this untoward feature. There-
fore, NST-732 can be used for detection of renal cell death,
as exemplified in the renal ischemia/reperfusion model pre-
sented in this study (Fig. 7).
While having a compact structure, NST-732 harbors both
a fluorophore, i.e., the dansyl group, and a fluorine atom,
potentially being an 18-F isotope, useful for imaging by
PET. The dansyl group, via its well-characterized fluores-
cent properties, allows detection of binding of NST-732 at
the single cell level. This allowed for cross-assessment of
cellular uptake of the compound versus binding of other
markers, for confirmation both in vitro and in vivo as to
the identity of the NST-732-binding cells as cells undergo-
ing cell death. It also provided elucidation of the correlation
between NST-732 binding and other molecular events in
the apoptotic cascade. These fluorescent properties demon-
strated the potential utility of NST-732 as detector of cell
death in several clinically-relevant animals models in vivo.
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2100 Apoptosis (2006) 11:2089–2101
Fig. 10 Uptake of NST-732 in vivo, into lymphoma tumor cells under-
going cell death in response to irradiation. Lymphoma (LY-S) tumors
were established in DBA mice. Mice were then treated by irradiation as
described in the text. NST-732 was administered intravenously 72 h af-
ter the last dose of irradiation. Two hours later, tumors were harvested
from control and irradiated mice, and processed as described under
Materials and Methods. Uptake of NST-732 (expressed as µg/g tumor
tissue) was quantified. As shown, average uptake of NST-732 in vivo
by the tumors wherein cell death was induced by irradiation (n =12)
was 12.5 fold higher than the uptake by the control untreated tumors
(n =8) (p<0.001)
Based on numerous lines of evidence, it is clear now that
apoptosis has a role in almost any medical disorder, either
in the etiology or pathogenesis of disease. Detection of cell
death in vivo may thus provide better diagnosis, monitoring
of disease course or assessment of treatment efficacy in these
various disorders. We chose to demonstrate this potential of
NST-732 in two major classes of applications: examples of
disorders in which apoptosis plays a role in the pathogenesis
of disease: renal ischemia/reperfusion, and cerebral stroke;
and a scenario wherein detection of cell death may assist in
assessment of the efficacy of therapy: monitoring of tumor
response to irradiation.
Ischemic or ischemia/reperfusion injury is very prevalent
in medical practice, andis amajor cause of organ dysfunction
and morbidity. Cell death in this clinical situation occurs both
in the ischemic and the reperfusion phases. In the model pre-
sented in our study, a relatively short-term ischemia (clamp-
ing of the renal artery for 45 min) was followed by a 24 h
period of reperfusion. Quite amazingly, even such a short
ischemic insult was found to be associated with a dramatic
process of cell death in the injured kidney. Such process can
explain renal failure after systemic hypotension or following
kidney transplantation. This load of cell death was well-
reported by NST-732, both at the whole-organ level and by
histopathology, and confirmed by correlation with TUNEL.
Detection of renal cell death by NST-732 may therefore have
important implications in early and more accurate diagnosis
or organ damage, and monitoring of effect of therapy. In that
aspect, ApoSense compounds such as NST-732 are unique,
as Annexin-V manifests very high non-specific binding to
the renal cortex, thus precluding its use for this purpose.
Cell death is also the major neuropathological substrate
in cerebral stroke, and recently, novel therapeutic strategies
that emerge for stroke management emphasize the need for
detection of the load of stroke-related cell death in order
to guide therapy. Thrombolytic therapeutic strategies, e.g.,
tissue plasminogen activators (tPA), offer a significant po-
tential therapeutic benefit, however with a substantial risk for
hemorrhagic complications. This raises an urgent need for
assessment of cerebral cell viability following the stroke. In
addition, emerging neuro-protective drugs, (e.g., NXY-059,
[23]) aiming at inhibiting brain cell death, also require mark-
ers for the process, for clinical assessment of their effect on
disease course. The study presents the potential usefulness
of NST-732 for this purpose. In a murine model of MCA
occlusion, systemic administration of NST-732 led to selec-
tive accumulation of the compound only in cells undergoing
cell death in the region of the infarct, culminating in a strong
signal, observed at the level of whole organ imaging.
A major current challenge in oncology is the lack of tools
for fast, non–invasive assessment of tumor response to anti-
cancer treatment. The concept of personalized medicine,
wherein treatment will be tailored according to the patient’s
genetic personal makeup, with optimization of therapy in
real-time as necessary, still awaits its implementation in clin-
ical oncology. At the level of the individual patient, the lack
of such tools is translated into frequent treatment failures,
and unnecessary exposure of the patient to the severe ad-
verse effects associated with anti-cancer treatments. At the
level of health providers, the current situation leads to subop-
timal allocation of therapeutic medical resources. Detection
of cell death can provide a direct report on treatment efficacy.
The study demonstrates the performance of NST-732 in this
context. In lymphoma-bearing mice subjected to irradiation,
we found that uptake of NST-732 was markedly increased
in response to treatment, respective of its accumulation in
the dying cells. Apoptosis is an important mode of cell death
induced by anti-cancer treatment, and thus its detection is
important for such clinical assessment. However, recent ev-
idence suggests a role also for other modes of cell death in
tumor response, such as necrosis, autophagy, mitotic catas-
trophe or senescence [24, 25]. Moreover, there is often a
continuum between features of the different modes of cell
death, and various modes of cell death can co-exist in a spe-
cific tumor in response to treatment. Importantly, membrane
alterations are prominent features of all these modes of cell
death, as assessed by Annexin-V and PI studies [24–26]. As
NST-732 targets the cell membrane, it is conceivable that
it may also be useful for detection of such modes of cell
death, extending beyond the classical apoptosis and necrosis
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Apoptosis (2006) 11:2089–2101 2101
dichotomy. Obviously however, further research is required
to evaluate NST-732 for this purpose.
Taken together, NST-732 administered intravenously in
vivo, selectively targeted cells undergoing cell death in vari-
ous clinically-relevant animal models, thus providing poten-
tially useful information on the extent of cell death associated
with the medical disorder or associated with treatment.
Based on these proof-of-concept studies, which support
the potential usefulness of NST-732 for imaging cell death
in clinical practice, we now proceed towards use of NST-
732 as a probe for PET. For this purpose we intend to use
the fluorine atom inherent in the molecule. Since
18
Fisre-
garded a preferred isotope for PET imaging due to its half-
life (110 min) and the signal that it provides, the synthetic
chemistry method to allow
18
F labeling of the compound
is now being developed. Such a method should allow rapid
and efficient attachment of the isotope in conditions com-
patible with routine clinical PET radio-chemistry. In view
of the possibility of introducing NST-732 as the first small-
molecule probe for clinical PET imaging of apoptosis, we
attach great importance to the results of the toxicological
studies performed, showing an excellent safety profile of the
compound as examined.
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