Mechanism of Retinoblastoma Tumor Cell Death after
Focal Chemotherapy, Radiation, and Vascular
Targeting Therapy in a Mouse Model
Maria-Elena Jockovich, Fernando Suarez, Armando Alegret, Yolanda Pin ˜a,
Brandy Hayden, Colleen Cebulla, William Feuer, and Timothy G. Murray
PURPOSE. To evaluate the mechanism and timing of retinal
tumor cell death in the LHBETATAGmouse model of retinoblas-
toma after treatment with vascular targeting therapies and
conventional therapies (focal chemotherapy and radiation).
METHODS. For vascular targeting therapy, 12- or 16-week-old
mice were treated with a single subconjunctival injection of
either anecortave acetate (300 ?g) or combretastatin A4 (1.5
mg). Eyes were analyzed at 1 day and 1 week after treatment.
Tumor cell death was evaluated using TUNEL assays or immu-
nofluorescence analysis of activated caspase 3 to detect apo-
ptosis. Histopathologic analysis was performed to identify ar-
eas of necrosis. For conventional therapy, LHBETATAGmice
were treated with six serial subconjunctival injections of fo-
cally delivered carboplatin chemotherapy (100 ?g/delivery) or
hyperfractionated external beam radiotherapy (EBRT; 15 Gy
total dose). Cell death was analyzed by TUNEL assay.
RESULTS. The highest levels of apoptotic cell death were seen 1
day after treatment in all treatment groups compared with
vehicle controls. At 1 week after treatment, apoptotic cell
death remained significantly elevated in the EBRT and carbo-
platin groups, but not after vessel targeting therapy. No signif-
icant necrosis was detected by histology in tumors of treated or
of control eyes.
CONCLUSIONS. Conventional therapies (focal carboplatin chemo-
therapy and EBRT) and vascular targeting agents significantly
increase cell death through apoptosis, while not having a
significant effect on necrosis in this murine model of retino-
blastoma. These studies will aid in the optimization of delivery
schemes of combined treatment modalities. (Invest Ophthal-
mol Vis Sci. 2007;48:5371–5376) DOI:10.1167/iovs.07-0708
16,600 live births in the United States.1Significant advances in
treatment have resulted in 5-year survival rates in Europe and
the United States of 90% and 98%, respectively.2,3
Tumor control and globe conservation with preservation of
sight have become the standard of care. Chemoreduction using
etinoblastoma, the most common intraocular tumor of
childhood, occurs in approximately 1 in 15,000 to 1 in
focal therapies have become a mainstay in the treatment of
moderate and large tumors. However, concerns regarding sec-
ondary malignancies still exist. Novel therapeutic strategies are
under investigation because of the morbidity and mortality
associated with current therapies. These strategies include
multimodality treatments using focally delivered chemother-
apy and vascular targeting therapy.
Retinoblastoma tumors are characterized by aggressive
growth, with continuing cellular proliferation occurring con-
comitantly along with cell death. Apoptosis, usually involved in
normal development and tissue repair, sometimes occurs spon-
taneously in these malignant tumors, markedly retarding their
growth.4–8Extensive necrosis is often found and is associated
with high-risk prognostic factors.9Analysis of retinoblastoma
cell death after treatment has been reported primarily in the
study of cell lines.10–14The induction of apoptosis is consid-
ered to be one of the principal mechanisms by which chemo-
therapy induces tumor regression.15Treatment using carbopla-
results in apoptotic cell death in
retinoblastoma cell cultures. Although these treatments result
in apoptotic cell death in vitro, studies of human retinoblas-
toma tumor samples after treatment have yielded inconclusive
results. Studies of retinoblastoma patient specimens do not
suggest a difference in apoptosis between treated and un-
treated groups.4The lack of difference in apoptosis between
these groups is likely attributed to the time lapse between
treatment and analysis, which in most studies has been 6
weeks. In fact, cell death by apoptosis occurs within 1 week of
Knowledge of the mechanism and timing of cell death after
individual treatments is essential for combined modality ther-
apy. However, it is not reasonable to analyze human retino-
blastoma samples at specific time points, and cell lines do not
fully replicate the intricate microenvironment of the intraocu-
lar tumor. Thus, animal modeling provides a way to optimize
treatment strategies and to study tumor cell death after ther-
Experiments using the LHBETATAGmouse model of retino-
blastoma suggest that combined treatment using carboplatin
and EBRT19or vascular targeting with anecortave acetate20
enables a reduction in the effective dose of carboplatin, reduc-
ing associated toxicities.20Combined therapy is more challeng-
ing than single modality therapy in that treatment scheduling
must be optimized to avoid adverse effects. Maximal treatment
response from a combined therapeutic approach will require a
highly coordinated dosing schedule that optimizes the timing
of induced tumor cell death in response to either therapy.
In this study, we investigated the mechanism of cell death
as a function of time after local vascular targeting agents
(anecortave acetate or combretastatin A4) and conventional
therapies (carboplatin chemotherapy or external beam radio-
therapy [EBRT]) to best formulate an approach for the timing
of combined therapeutic modalities in the future.
From the Department of Ophthalmology, Bascom Palmer Eye
Institute, University of Miami School of Medicine, Miami, Florida.
Supported by National Institutes of Health Grant R01 EY013629
and Center Grant P30 EY014801, and by an unrestricted grant to the
University of Miami from Research to Prevent Blindness, Inc.
Submitted for publication June 12, 2007; revised August 24, 2007;
accepted October 17, 2007.
Disclosure: M.-E. Jockovich, None; F. Suarez, None; A. Alegret,
None; Y. Pin ˜a, None; B. Hayden, None; C. Cebulla, None; W. Feuer,
None; T.G. Murray, None
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked “advertise-
ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Timothy G. Murray, Department of Oph-
thalmology, Bascom Palmer Eye Institute, P.O. Box 016880, Miami, FL
Investigative Ophthalmology & Visual Science, December 2007, Vol. 48, No. 12
Copyright © Association for Research in Vision and Ophthalmology
This study protocol was approved by the University of Miami Animal
Care and Use Committee. All experiments in this study were con-
ducted in accordance with the ARVO Statement for the Use of Animals
in Ophthalmic and Vision Research.
The LHBETATAGtransgenic mouse model used in this study has been
characterized previously.21Presence of the SV40Tag was detected by
PCR analysis of tail biopsies. To detect apoptosis during tumor pro-
gression, retinal tumors from LHBETATAGmice of 4, 8, 12, and 16 weeks
of age were analyzed (n ? 5 per treatment group). LHBETATAGmice
typically develop microscopic tumors by age 4 weeks, small tumors by
age 8 weeks, medium tumors by age 12 weeks, and large tumors that
often fill the available globe space by age 16 weeks.22
Subconjunctival Injections and Drug Delivery
Injections were delivered with a 33-gauge needle inserted into the
superotemporal subconjunctival space. LHBETATAGmice of either 12
(n ? 5 per group) or 16 (n ? 5 per group) weeks of age received a
single subconjunctival injection of anecortave acetate or combretasta-
tin A 4P (CA4P). Drug doses that are known to result in the highest
reduction of tumor burden were chosen for this study.20,23,24Anecor-
tave acetate (Alcon Pharmaceuticals, Forth Worth, TX) was delivered at
a dose of 300 ?g/20 ?L; CA4P (OxiGene, Inc, Watertown, MA) was
given at a dose of 1.5 mg/20 ?L Carboplatin was delivered to 10-week-
old LHBETATAGmice (n ? 6 per group) by six serial, biweekly subcon-
junctival injections at a dose of 100 ?g/delivery. Mice were humanely
killed, and enucleations were performed at 1 day, 1 week, and 4 weeks
after the termination of treatment.
Ten-week-old LHBETATAGmice (n ? 6 per group) received EBRT in 120
cGy fractions delivered twice daily at a 6-hour interfraction interval for
a total dose of 15 Gy (10 mV; Clinac 2100; Varian Medical Systems,
Inc., Palo Alto, CA). Radiation dose and delivery schedule were chosen
to obtain optimal response.25Mice were humanely killed 1 day, 1
week, and 4 weeks after termination of the last treatment. Eyes were
fixed, paraffin embedded, and serially sectioned. Six sections per
sample were obtained. The number of labeled cells in a ?400 field was
Detection of Apoptotic Cells
For the detection of apoptotic cells during tumor progression and after
vessel targeting therapy, eyes were embedded in optimum cutting
temperature compound, snap frozen in liquid nitrogen, and sectioned
(8 ?m). Sections were fixed with fresh 4% paraformaldehyde. Apopto-
sis detection was performed with a TUNEL based-kit (ApopTag Fluo-
rescein In Situ Apoptosis Detection Kit; Chemicon, Temecula, CA) that
detects apoptosis by adding digoxigenin nucleotides to the 3?-OH ends
of double or single-stranded DNA. An anti–digoxigenin antibody con-
jugated with fluorescein is then added and followed by DAPI antifade
(Chemicon) for immunofluorescence detection. Only cells that colo-
calized with DAPI fluorescence were counted as positive cells. Apo-
ptotic cell death was also detected with a rabbit antiactivated caspase
3 (AC3) antibody (1:100; Chemicon). Goat anti-rabbit fluorescent dye
(Alexa Fluor 568; 1:500; Invitrogen, Carlsbad, CA) was used as a
secondary antibody. Vascular endothelial cells were detected with
fluorescent dye–conjugated lectin (Bandera simplicifolia; Alexa Fluor
568; 1:1000; Invitrogen). The ratio of endothelial cell death to apopto-
tic cell death was given as the number of apoptotic cells (as deter-
mined with AC3) that colocalized with lectin to the total number of
Serial cross-sections of eyes containing tumors were examined for
the presence of the different markers with an upright fluorescence
microscope (BX51; Olympus America Inc., Melville, NY). All images
were digitally acquired and recompiled (Photoshop CS; Adobe, San
Jose, CA). Sections were viewed at ?40 magnification.
For the detection of apoptosis after carboplatin treatment or EBRT,
eyes were analyzed at 1 day, 1 week, and 4 weeks after termination of
the last treatment. Eyes were fixed with 10% formalin and embedded
in paraffin. In situ detection of apoptotic cells was conducted using the
anti–BRDU antibody and a detection kit (TACS; R&D Systems, Minne-
apolis, MN). Labeled cells in ?400 fields were counted. At least three
fields were counted and averaged.
Unless otherwise specified, data were analyzed according to analysis of
variance (ANOVA). Post hoc tests were performed by the least signif-
icant difference test.
Tumor Cell Death during Tumor Progression
To assess cell death during tumor development, retinal tumors
from LHBETATAGmice of 4, 8, 12, and 16 weeks of age were
analyzed for apoptosis using both the TUNEL assay and a
marker for AC3. In LHBETATAGmice, cell death was not de-
tected in small tumors (4–8 weeks of age). In larger tumors,
low levels of apoptosis were detected (2–3 per high-power
field; ?400; Fig. 1). The highest numbers of apoptotic cells
were seen in retinal tumors from 12-week-old mice. The levels
of apoptosis detected by the TUNEL assay at 12 weeks of age
appeared higher than those detected by AC3. However, this
difference was not statistically significant (P ? 0.133). Sixteen-
week-old mice had a mean number of apoptotic cells that was
significantly lower than the mean number of apoptotic cells in
12-week-old mice (P ? 0.002, TUNEL; P ? 0.052, AC3). Ne-
crosis was not detected by histology in these eyes (data not
Tumor Cell Death after Vessel Targeting Therapy
To assess cell death after vessel targeting therapy, 16-week-old
LHBETATAGmice (n ? 5 per group) were treated with a single
subconjunctival injection of either anecortave acetate (300 ?g)
or CA4P (1.5 mg). Tumors were analyzed for apoptosis or
necrosis 1 day or 1 week after treatment. Both TUNEL assay
and AC3 immunohistochemistry yielded the same results.
There was significantly higher apoptotic cell death 1 day after
vessel targeting therapy with either agent than the untreated
age-matched controls (P ? 0.001; both, Fig. 2). The data
further suggest a differential induction of tumor cell death
relative to time of treatment and vascular targeting agent. The
highest amount of apoptotic cell death was detected with both
treatments 1 day after injection. The number of apoptotic cells
in these advanced tumors decreased significantly by 1 week
after treatment (P ? 0.002, anecortave acetate; P ? 0.001,
CA4P). Statistical analysis of AC3 data suggests that the amount
of apoptotic cell death varies with treatment. The two drugs/
two time points and controls were compared in a one-way
ANOVA. The average count of all fields was examined for each
animal, and the average apoptotic cell count of all fields was
examined for each animal. Average numbers of apoptotic cells
were as follows: no treatment, 1.8; 1 day anecortave acetate,
11.3; 1 week anecortave acetate, 3.5; 1 day CA4P, 25.5; 1 week
CA4P, 3.5. The differences in apoptotic cell count between
treatment groups 1 day after injection, between treated groups
1 day after injection relative to untreated controls, and be-
tween 1 day and one 1 week time points after treatment with
each drug were highly significant (P ? 0.001). There was no
significant difference between the two 1-week posttreatment
groups (Fig. 2).
5372 Jockovich et al.
IOVS, December 2007, Vol. 48, No. 12
Endothelial Cell Death after
To assess the amount of endothelial cell death after vessel-
targeting therapy, LHBETATAGmice were treated at 12 or 16
weeks of age with anecortave acetate or CA4P. Eyes were
analyzed by immunohistochemical analysis for AC3 and lectin
at 1 day and 1 week after treatment. Our data show that only
approximately 50% of total apoptotic cells after treatment with
anecortave acetate or CA4P are endothelial cells (Fig. 3). En-
dothelial to total apoptotic cell ratio is significantly higher in
treated animals than in untreated littermate controls at 12
weeks (P ? 0.004, two-sample t-test) and 16 weeks (P ? 0.005,
two-sample t-test). The mean ratio of endothelial cell death to
total cell death in the 12-week treatment groups was 0.560 ?
0.26 (? SE); at 16 weeks of age, the mean was 0.453 ? 0.21
(? SE). Further analysis by three-factor ANOVA showed that
there was a significant difference (P ? 0.003) in endothelial
cell death between the 12-week and the 16-week age groups
but not between treatment agents (P ? 0.76) or days after
treatment (P ? 0.87). These results suggest that vascular tar-
geting therapy is more effective in promoting endothelial cell
apoptosis when mice are treated at 12 weeks of age than when
they are treated at 16 weeks of age. This difference appeared to
be more evident when eyes were analyzed 1 day after anecor-
tave acetate treatment. Necrosis was not detected in these eyes
after vessel targeting therapy with either agent at either time
point (not shown).
Tumor Cell Death in Response to
To assess cell death after either radiotherapy or carboplatin
chemotherapy, 10-week-old mice received carboplatin or EBRT
as described in Methods. Eyes were analyzed 1 day, 1 week,
and 4 weeks after treatment completion (Fig. 4). At day 1, eyes
treated with 100 ?g carboplatin chemotherapy exhibited a
10.6-fold increase in the percentage of apoptotic cells over
control eyes. This increased slightly at 1 week to 12.1-fold. By
4 weeks after treatment, the levels of apoptotic cell death were
down to those seen in untreated controls. Eyes treated with
EBRT at a dose of 15 Gy also exhibited an increase in apoptotic
cell death compared with the control. There was a 10.3%
increase at day 1, a 4.4% increase at 1 week, and no significant
difference at 4 weeks relative to the control samples. Control
eyes in this model revealed approximately a 1% incidence of
cell death. Bars represent number of
apoptotic cells per high-power field
(HPF; ?400) marked with TUNEL
(■), or activated caspase 3 (■). Error
bars represent SD from the mean
(n ? 5). The difference in mean ap-
optotic numbers measured by TUNEL
and activated caspase 3 is not statis-
tically significant (P ? 0.13).
Natural history of tumor
vessel targeting therapy. LHBETATAG
mice of 16 weeks of age were
treated with single subconjunctival
injection of either anecortave ace-
tate (300 ?g) or CA4P (1.5 mg). Bars
represent number of apoptotic cells
per high-power field (HPF; ?400)
marked with TUNEL (■) or activated
caspase 3 (■). Error bars represent
SD from the mean (n ? 5).
Tumor cell death after
IOVS, December 2007, Vol. 48, No. 12
Retinoblastoma Tumor Cell Death5373
cells in apoptosis throughout the time points examined. The
percentage of cells in apoptosis ranged from 0.6% at 1 week to
1.8% at 4 weeks. No statistical difference was noted at any time
point within these control eyes. These results show that at 1
day and 1 week, both the carboplatin and the EBRT groups
have a significantly greater incidence of apoptotic cell death
than the control (P ? 0.001 and P ? 0.02, respectively). At 1
week after treatment, carboplatin-treated tumors had a signif-
icantly greater percentage of cells in apoptosis than EBRT-
treated tumors (P ? 0.007) and untreated controls (P ? 0.001).
At this time point, EBRT-treated tumors also had significantly
higher levels of apoptosis than untreated controls (P ? 0.037).
The levels of apoptosis had decreased by 4 weeks after both
Novel therapeutic strategies for the treatment of retinoblas-
toma optimally involve targeted timed multimodality ap-
proaches for tumor control. Optimal combined therapies re-
quire an understanding of the pharmacokinetics of each
individual drug and the mechanism and timing of cell death
after individual treatments. In this study, the timing and mech-
anism of retinal tumor cell death were investigated after treat-
ment with commonly used therapeutics (carboplatin and radi-
ation) and the novel antiangiogenic and angiostatic therapeutic
strategies under investigation. Data from this study suggest that
apoptosis is the mechanism for cell death in the LHBETATAG
mouse model of retinoblastoma undergoing radiotherapy, focal
endothelial cells relative to total ap-
optotic cells after vessel targeting
therapy in mice of 12 weeks and 16
weeks of age. (A) Representative mi-
crograph illustrating apoptotic cells
and endothelial cells 1 day after
treatment with anecortave acetate.
Apoptotic cells stained with acti-
vated caspase 3 (A), endothelial cells
stained with lectin (B), colocaliza-
tion (C), and DAPI nuclear stain (D).
(B) Bars represent percentage of ap-
optotic cells that colocalized with
endothelial cells to total apoptotic
cells per high-power field in animals
treated at 12 weeks (■) and 16
weeks (■) of age. Error bars repre-
sent SD from the mean (n ? 5).
Percentage of apoptotic
5374 Jockovich et al.
IOVS, December 2007, Vol. 48, No. 12
chemotherapy, or vessel-targeting therapy. Differential induc-
tion of apoptosis is noted after treatment with early enhance-
ment associated with radiotherapy and vessel targeting therapy
and late enhancement with chemotherapy.
Vascular targeting with antiangiogenic and angiostatic
agents is emerging as a possible treatment option for retino-
blastoma given the tumor’s dependence on vascular supply
and its potential to promote angiogenesis. We demonstrated
that two different vessel-targeting agents (combretastatin A4
and anecortave acetate) effectively reduce tumor burden in the
LHBETATAGmodel of retinoblastoma.20,23,26Our previous work
also found that when anecortave acetate is used together with
carboplatin, the dose and delivery schedule must be optimized
to avoid adverse effects.20Data from the present study suggest
that both agents induce rapid caspase-dependent apoptotic cell
death within 1 day of treatment.
A higher percentage of endothelial cell death to total cell
death was detected in mice treated at 12 weeks of age than in
mice treated at 16 weeks of age. A plausible explanation for
this finding is that younger mice have a higher percentage of
angiogenic vasculature than older mice. We have recently
characterized blood vessel maturation in the LHBETATAGmouse
model of retinoblastoma.22Angiogenesis in developing retinal
tumors was detected in the early stages of tumor development
and increased with age, decreasing slightly in advanced dis-
ease. On the other hand, tumor vessel maturation does not
occur until advanced disease develops at 12 to 16 weeks of
age; the amount of pericyte-committed vasculature increases
with age. Endothelial cells in newly formed vessels require
growth factors for survival; in their absence, such as after
antiangiogenic treatment, the endothelial cells undergo apo-
ptosis and regress.27Mature vessels are stabilized by pericytes
and are no longer dependent on angiogenic stimuli, thus, they
may be resistant to antiangiogenic treatment. We have re-
ported that treatment with vessel-targeting agents CA4P and
anecortave acetate in advanced disease, though reducing total
numbers of endothelial cells, did not effectively target mature
vasculature.22Results from this study suggest that vascular
targeting is more effective in the treatment of small tumors
harbored by younger animals and may have restricted efficacy
in the treatment of large tumors, limiting the clinical efficacy of
Antiangiogenic and cytotoxic chemotherapy potentially
yield maximal effects when combined because different cells
in the tumor mass are targeted cancer cells and endothelial
cells.28Targeting vasculature, however, may compromise the
delivery of chemotherapy to the tumor and may antagonize the
effect of the combined therapy.29Studies have shown that
endothelial cell apoptosis precedes tumor cell death in many
solid tumors.30The premise behind the use of antiangiogenic
therapy for solid tumors is that though it kills endothelial cells
that feed the tumor, causing tumor cell death, these capillaries
are also responsible for delivering chemotherapeutic drugs to
the tumor. Thus, vessel targeting may inhibit coincident drug
delivery. We have previously shown that the greatest reduction
of tumor burden is achieved when anecortave acetate treat-
ment follows a cycle of six carboplatin injections; if anecortave
acetate is given during the carboplatin cycle, then the syn-
ergistic effect of the combination of the two drugs is lost.20
Based on the data from the present study and our previous
results, we believe that an ideal dosing regimen would be six
cycles of carboplatin chemotherapy followed 1 week later
by a single injection of anecortave acetate. This delivery
scheme would presumably increase tumor cell death while
minimizing toxic adverse effects associated with high doses
The vessel-targeting agents used have different mechanisms
of action: anecortave acetate is an angiostatic cortisene that
prevents new endothelial blood vessel formation by inhib-
iting growth factors required for endothelial cell survival.31
CA4P induces endothelial cell death by arresting cells in
mitosis.32The ensuing vessel collapse results in a rapid
ischemic necrotic cell death in surrounding tumor cells.
Interestingly, we detected the same type of cell death after
treatment with either drug. No signs of necrosis were detected
in these tumors.
In conclusion, this study demonstrates that apoptosis is a
mechanism for cell death in the LHBETATAGmouse model of
retinoblastoma undergoing radiotherapy, subconjunctival che-
motherapy, or vessel-targeting therapy. Further, we have
shown that there is a differential timing of the induction of
apoptosis after different treatment modalities. This differential
timing of induction of apoptosis may account for synergistic
therapeutic interactions noted during combined treatment and
may have implications in combined modality therapies for
after chemotherapy and EBRT.
LHBETATAGmice of 10 weeks of
age were treated with six bi-
weekly subconjunctival injections
of carboplatin (100 ?g/delivery)
or EBRT (120 cGy fractions for a
total dose of 15 Gy). Bars repre-
sent percentage of apoptotic cells
in untreated controls, (■), after
treatment with carboplatin (■), or
EBRT (■). Error bars represent SD
from the mean (n ? 6).
IOVS, December 2007, Vol. 48, No. 12
Retinoblastoma Tumor Cell Death5375
Acknowledgments Download full-text
The authors thank Amy Schefler for careful review of the manuscript
and Magda Celdran for excellent histologic examination.
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