Download Effectiveness of the Angiogenesis Inhibitor TNP

Vol. 5, 4273– 4278, December 1999
Clinical Cancer Research 4273
Effectiveness of the Angiogenesis Inhibitor TNP-470 in Reducing the
Growth of Human Neuroblastoma in Nude Mice Inversely
Correlates with Tumor Burden1
Howard M. Katzenstein, Alfred W. Rademaker,
Christof Senger, Helen R. Salwen,
Nadine N. Nguyen, Paul S. Thorner, Louis Litsas,
and Susan L. Cohn2
Departments of Pediatrics [H. M. K., S. L. C.] and Pathology [C. S.],
Children’s Memorial Hospital, Chicago, Illinois 60614; Robert H.
Lurie Comprehensive Cancer Center, Northwestern University
Medical School, Chicago, Illinois 60611 [H. M. K., S. L. C.,
A. W. R., H. R. S., N. N. N.]; and Department of Pediatric Laboratory
Medicine, Division of Pathology, Hospital for Sick Children and
University of Toronto, Toronto, MSG 1X8 Canada [P. S. T., L. L.]
evident small (<400 mm3; n 5 15) and large (>400 mm3;
n 5 11) tumors. For animals with small tumors, the mean
rate of growth was significantly decreased in the treated
mice compared to the controls (P 5 0.02). In contrast, there
was no difference in the mean rate of tumor growth between
animals with large tumors treated with TNP-470 and controls (P 5 0.64). Our studies demonstrate that the effectiveness of TNP-470 inversely correlates with tumor burden. We
speculate that TNP-470 may most effectively inhibit NB
tumor growth in children with a low tumor burden.
INTRODUCTION
ABSTRACT
Angiogenesis plays an important role in the growth and
metastasis of malignant tumors. We have previously reported that in children with neuroblastoma (NB), tumor
vascularity directly correlates with metastatic disease,
MYCN amplification, and poor outcome. The angiogenesis
inhibitor TNP-470 has been shown to reduce the rate of NB
growth in rodents with macroscopic tumors without ultimately impacting survival. To investigate whether TNP-470
could more effectively inhibit NB growth in animals with a
low tumor burden, we treated 30 nude mice with minimal
disease with this angiogenesis inhibitor (supplied by TAP
Pharmaceuticals, Inc.). Therapy was initiated before tumors
were clinically evident after s.c. inoculation of 5 3 106 cells
from the MYCN-amplified NB cell line NBL-W-N. TNP-470
was administered 3 days/week, and after 12 weeks of treatment, 53% of the treated mice remained tumor free,
whereas 100% of the control mice developed tumors (P <
0.0001). To further assess the relationship between the efficacy of TNP-470 treatment and tumor burden, TNP-470 was
also administered s.c., 3 days/week, to mice with clinically
Received 6/22/99; revised 9/13/99; accepted 9/13/99.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
1
Supported in part by the National Cancer Institute, NIH Grant
CA74824 (to S. L. C.), the Elise Anderson Neuroblastoma Research
Fund (S. L. C.), the Michelle Carroll Research Fund (S. L. C.) the
Neuroblastoma Children’s Cancer Society (S. L. C.), Bear Necessities
Pediatric Cancer Foundation (H. M. K.), Bears Care (S. L. C.), VisionTek Foundation (S. L. C.), gifts from Ezra Schaffer and Dennis Drescher (to S. L. C.), and the Robert H. Lurie Comprehensive Cancer
Center NIH National Cancer Institute Core Grant 5P30CA60553.
2
To whom requests for reprints should be addressed, at Children’s
Memorial Hospital, Division of Hematology/Oncology, Box 30, 2300
Children’s Plaza, Chicago, IL 60614. Phone: (773) 880-4562; Fax:
(773) 880-3223; E-mail: [email protected].
Sustained growth and metastases of solid tumors are dependent on the development of an adequate blood supply
through angiogenesis (1, 2). This process is mediated by an
imbalance of angiogenic inducers and inhibitors released from
both neoplastic cells and host cells (1–3). Numerous studies
have demonstrated that outcome in adults with a variety of
malignancies can be predicted by the degree of tumor angiogenesis (4 – 8). We previously reported that high tumor vascularity was associated with MYCN amplification, advanced-stage
disease, and poor outcome in children with NB3 (9). Conversely,
excellent survival was observed in the subset of children included in our analysis who had tumors with low vascular density. These observations suggest that high levels of angiogenic
activators are likely to be present in NB tumors that are MYCNamplified and rapidly progressive and that agents capable of
interfering with the process of angiogenesis may inhibit or slow
NB tumor growth. Thus, angiogenesis inhibitors may prove to
be an effective modality of therapy for children with highly
vascular, clinically aggressive NB tumors.
TNP-470, O-(N-chloroacetyl-carbamoyl)-fumagillol, a
semisynthetic analogue of fumagillin, is a potent inhibitor of
angiogenesis (10). The antitumor effect of TNP-470 has been
analyzed in a variety of experimental animal models, (11–14),
and TNP-470 has recently been evaluated in Phase I and Phase
II clinical trials (14, 15). In preclinical NB studies, TNP-470
treatment reduced the rate of primary tumor growth in tumorbearing mice compared to control treatment. In addition, a
decreased incidence of metastasis to the axillary lymph nodes
and the liver was observed with TNP-470 treatment in one of the
murine NB models. However, tumors continued to grow in the
animals, and all mice eventually died from tumor progression.
To investigate whether TNP-470 would be more effective
in the setting of minimal disease, we initiated treatment in nude
mice before tumors were clinically apparent, either 12 h or 1
3
The abbreviations used are: NB, neuroblastoma; MKI, mitotic-karyorrhexis index.
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4274 Angiogenesis Inhibition in Neuroblastoma
week after the mice were s.c. inoculated with cells from the
human NB cell line NBL-W-N (16). We also treated animals
3–9 weeks after tumor cell inoculation, once small (,400 mm3)
or large (.400 mm3) tumors developed, to evaluate whether the
effect of TNP-470 treatment differed according to tumor burden.
Our studies demonstrate that TNP-470 treatment does effectively inhibit NB growth when the agent is administered in the
setting of minimal disease. Furthermore, when TNP-470 is
administered to animals with small tumors, the rate of growth is
reduced. However, TNP-470 treatment does not significantly
alter the tumor growth rate when it is administered to animals
with large tumors.
MATERIALS AND METHODS
Cell Culture. The MYCN-amplified human NB cell line
NBL-W-N, a subclone of the NBL-W cell line, was established
in our laboratory and has been described previously (16). The
cells were grown in DMEM supplemented with 10% heatinactivated fetal bovine serum plus antibiotics and glutamine
(Life Technologies, Inc., Gaithersburg, MD).
TNP-470. TNP-470, a generous gift of TAP Pharmaceuticals, Inc. (Deerfield, IL), was stored at 4°C. A 10% stock
solution of TNP-470 in ethanol was made weekly. For the
animal studies, the stock solution was diluted 1:70 or 1:35 with
PBS immediately before injection (Life Technologies, Inc.).
Animal Studies. Male 4 – 6-week old homozygous athymic nude mice were obtained from Harlan Sprague Dawley
(Madison, WI). The mice were housed in a laminar flow caging
system, fed ad libitum, and treated in accordance with a protocol
approved by the Northwestern University Animal Care and Use
Committee. All mice were inoculated s.c. with 5 3 106 NBLW-N cells in the right flank.
A total of 30 mice with minimal disease received TNP-470
(30 mg/kg) s.c. at a distant site in three different experiments,
beginning either 12 h (n 5 21) or 1 week (n 5 9) after tumor cell
inoculation, before tumors were clinically evident. Subsequently, the mice were treated with TNP-470 (7.5 mg/kg) twice
daily on Monday, Wednesday, and Friday for 12 weeks. To
investigate whether prolonged administration of TNP-470
would enhance survival, a subgroup of four mice received
prolonged treatment with TNP-470 for a total of 17 weeks. Mice
were observed for a total of 12 months after tumor cell inoculation and then euthanized. Control mice (n 5 16) received
injections at the same intervals with vehicle alone (1% ethanol
in PBS).
In separate studies of mice with macroscopic disease, TNP470 (30 mg/kg) was administered s.c. once daily on Monday,
Wednesday, and Friday to mice (n 5 15) with clinically evident
small tumors (defined as ,400 mm3) and mice (n 5 11) with
large tumors (.400 mm3). At the same time, 13 control mice
with large (n 5 5) or small (n 5 8) tumors were treated with
vehicle alone. Tumor volume was measured weekly using the
formula tumor volume 5 (length 3 width2)/2 (17). Mice were
sacrificed at the first sign of illness or when they had clinical
evidence of discomfort from the tumor.
Proliferation and Cytotoxicity Assays. NBL-W-N cells
were seeded into 96-well tissue culture plates at a concentration
of 5 3 103 cells/well in complete DMEM. After the cells were
incubated for 24 h at 37°C, test media containing TNP-470 in
concentrations ranging from 1 mg/ml (2.5 mM) to 1 pg/ml (2.5
pM) or control media containing 1% ethanol were added to the
wells. The cells were refed 48 h later with fresh test media and
incubated at 37°C for an additional 24 h, and the number of
proliferating cells was then measured using the CellTiter 96 AQ
nonradioactive cell proliferation assay (Promega Corp., Madison, WI). The number of proliferating cells in the cultures
containing TNP-470 was compared to the number of cells
cultured in control media (100%). All conditions were assayed
in quadruplicate. Similar conditions were used to assess the
cytotoxic effects of TNP-470 on the NBL-W-N cells. The number of surviving cells cultured in media containing TNP-470 in
concentrations ranging from 1 mg/ml (2.5 mM) to 1 mg/ml (2.5
mM) was determined using trypan blue exclusion and compared
to the number of surviving cells cultured in the control media.
Histopathology and Immunohistochemistry. Tumor
tissue for histological examination was obtained from moribund
animals immediately after they were sacrificed. In addition,
tumor tissue was processed for histology in a small number of
control and treated mice sacrificed before they exhibited signs
of illness, after 2 weeks of treatment. Tumor tissue was fixed in
10% buffered formalin and embedded in paraffin. Four-mmthick histological sections were cut for H&E staining. Histological analyses were performed without knowledge of animal
treatment. The MKI was calculated for each case according to
the criteria described by Shimada et al. (18). The percentage of
necrosis was estimated after examining at least ten 3400 fields.
Determination of Microvessel Density. To highlight
endothelial cells, immunohistochemical staining with mouse
monoclonal anti-CD31 antibody (Cell Marque, Austin, TX) was
performed on additional formalin-fixed and paraffin-embedded
tumor sections using an automated stainer (Ventana, NEXES,
Tucson, AZ) according to the manufacturer’s instructions. Microvessel number was assessed in tumor areas showing the
highest density of staining as determined by an initial scan with
low magnification (320), as described previously (5). For vessel counting, a 3200 field in each of three to four of the most
vascular areas was counted, and the average counts were recorded. Structures clearly identifiable as vessel or vascular buds
(vessels without a clear vascular lumen) were included in the
count. Vessels with a diameter of .50 mm were excluded.
Vessels in fibrovascular septa were counted only once if they
were identified as the same vessel. Vessel counting was performed without knowledge of the treatment the animal received.
Apoptosis Analysis. Determination of apoptotis in situ
was performed based on the terminal deoxynucleotidyl transferase-mediated nick end labeling assay using the ApopTag Kit
(Intergren Company, Purchase, NY) according to the manufacturer’s directions. Stained slides were examined on a Leitz
Dialux (Leica Canada) photomicroscope with a color video
camera (Pulnix TMC-7; Bock Optronic, Inc.) connected to it.
The video camera was attached to a personal computer Pentium
II (Window’s NT 95). A video image of the slide showing
positive and negative nuclei was created on the computer monitor using the ATI Version 4.35 video imaging program using a
310 ocular and a 325 objective. The video image was captured
and analyzed using Scion Image 1998 software (Scion Corp.).
Positive and negative nuclei were scored separately. A total of
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Clinical Cancer Research 4275
five different fields were captured from each slide. To determine
the percentage of apoptotic nuclei for a specific sample, the total
number of positive nuclei was divided by the total number of
nuclei (both positive and negative) and multiplied by 100.
Statistical Analysis. Tumor-free survival of the treated
and control groups with minimal disease was compared by
GraphPad Prism software (GraphPad Software, San Diego, CA)
using the log-rank test. Because small numbers of mice were
sacrificed for histological studies after 2 weeks of treatment,
tumor volumes were determined using the last value carried
forward when necessary. Tumor volume, tumor growth rate,
MKI, blood vessel number, and tumor necrosis were analyzed
using the independent sample t test to compare control and
treatment groups stratified according to large or small tumor
size (19).
RESULTS
In Vivo Tumor Growth. To investigate whether TNP470 would effectively inhibit NB growth in animals with minimal tumor burden, 46 mice were inoculated with NB cells and
treated with either TNP-470 or control vehicle before the emergence of clinically palpable tumors. After 12 weeks of treatment, 16 of 30 (53%) mice treated with TNP-470 were tumor
free, and 23 (77%) mice were alive. Over a median follow-up of
11 months (range, 4 –12 months), 14 of the TNP-470-treated
mice remained disease free, 13 mice died from local tumor
progression, and 3 mice died secondary to toxicity, including
two mice from the subgroup that received prolonged TNP-470
therapy. There was no statistical difference in tumor-free survival between the animals treated 12 h after tumor inoculation
and those initially treated at 1 week (P 5 0.75). In contrast,
within the same 12-week period of time, tumors developed in all
16 (100%) control mice, and 15 (94%) animals died from tumor
progression (P , 0.0001; Fig. 1).
To further investigate the relationship between tumor burden and the effectiveness of TNP-470 treatment in retarding NB
growth, treatment was also administered to mice with clinically
apparent small (,400 mm3) or large (.400 mm3) tumors. A
significant decrease in mean tumor growth rate was observed
when TNP-470 was administered to mice bearing small tumors
compared to that seen in the control animals (36 6 8 mm3/day
versus 98 6 24 mm3/day, respectively; P 5 0.02; Table 1). In
addition, after 3 weeks of treatment with TNP-470, the average
tumor volume in mice with small tumors was significantly lower
than that observed in controls (P 5 0.02; Fig. 2A). In contrast,
the mean rate of tumor growth in the animals with large tumors
treated with TNP-470 did not differ significantly from that seen
in the control animals bearing large tumors (259 6 47 mm3/day
versus 220 6 69 mm3/day respectively; P 5 0.64). Similarly,
after 2 weeks of therapy, the average tumor volume in TNP470-treated mice with large tumors did not differ significantly
from that of controls (P 5 0.93; Fig. 2B.). In this model, none
of the animals developed metastatic disease.
Histology. The xenografts from both the treated and control animals were composed of sheets of neuroblasts with scant
cytoplasm (Fig. 3). There was almost no connective tissue and
no evidence of Schwannian stroma or neuropil. A small number
of neuroblasts exhibited some evidence of morphological dif-
Fig. 1 Tumor-free survival of nude mice treated for 12 weeks with
either TNP-470 (n 5 30) or control vehicle alone (n 5 16; treatment was
initiated before the tumors were clinically apparent). Tumor-free survival was compared using the log-rank test. The number of mice at risk
is shown in parentheses.
ferentiation with increased amounts of cytoplasm and vesicular
nuclei. However, complete ganglionic differentiation was not
observed. The mean MKI was similar in each of the subgroups
analyzed (Table 1). There was no significant difference in the
number of apoptotic cells or in the percentage of necrosis
detected in the tumors from the animals treated with TNP-470
versus control vehicle. Similarly, the number of vessels counted
in the treated tumors was not significantly different from that
observed in the control tumors.
Toxicity. Treatment was generally well tolerated in the
animals treated with TNP-470 at a dose of 30 mg/kg/day, three
times/week. During the 12-week treatment regimen, 4 of 30
mice developed cachexia and weight loss, which resolved when
the drug was discontinued. To discern whether continued therapy would result in a further improvement in survival, a subgroup of four mice continued to receive treatment for a total of
17 weeks. However, therapy was discontinued when similar but
irreversible toxicity occurred in two of the four mice. In addition, one of the treated mice developed a palpable tumor 8
weeks after tumor inoculation and died days later of a pulmonary hemorrhage.
In Vitro Proliferation and Cytotoxicity Studies. In
vitro proliferation and cytotoxicity assays were performed to
determine whether the antitumor effect of TNP-470 was angiostatic, cytostatic, and/or cytotoxic. NBL-W-N cell proliferation
was not affected by TNP-470 in concentrations ranging from 1
pg/ml to 1 mg/ml. However, in the presence of 10 mg/ml (25
mM) TNP-470, NBL-W-N cell proliferation was inhibited by
16% compared to control. Proliferation was completely blocked
at TNP-470 concentrations of 100 mg/ml (250 mM; Fig. 4).
Cytotoxicity of the NBL-W-N cells was first observed at TNP470 concentrations of 100 mg/ml (250 mM; data not shown).
DISCUSSION
This study demonstrates that the effectiveness of the angiogenesis inhibitor TNP-470 is inversely related to NB tumor
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4276 Angiogenesis Inhibition in Neuroblastoma
Table 1
Effect of TNP-470 on the growth rate, histopathology, and vascularity of NB xenografts
Small tumors (,400 mm3)
3
Rate of tumor growth (mm /day)
MKI/5000 cells
No. of blood vessels/high-power field
Tumor necrosis (%)
a
Controls
TNP-470treated mice
Mean 6 SE (n)
Mean 6 SE (n)
98 6 24 (8)
154 6 9 (7)
61 6 6 (7)
12 6 5 (7)
36 6 8 (15)
158 6 21 (12)
67 6 9 (9)
12 6 4 (12)
Large tumors (.400 mm3)
Controls
TNP-470treated mice
P
Mean 6 SE (n)
Mean 6 SE (n)
P
0.02
NS
NS
NS
220 6 69 (5)
152 6 31 (3)
63 6 3 (3)
18 6 11 (3)
259 6 47 (11)
166 6 12 (7)
62 6 6 (7)
3 6 1 (7)
NSa
NS
NS
NS
NS, not significant.
Fig. 2 A, tumor growth of NB xenografts in animals bearing small
tumors (,400 mm3) treated with TNP-470 (n 5 15) versus control
vehicle (n 5 8). The mean 6 SE of tumor volume is shown. B, tumor
growth of NB xenografts in mice bearing large tumors (.400 mm3)
treated with TNP-470 (n 5 11) versus control vehicle (n 5 5). The
mean 6 SE of tumor volume is shown.
burden. TNP-470 was administered to three groups of mice after
s.c. inoculation with cells from the MYCN-amplified human NB
cell line NBL-W-N (16). NBL-W-N cells were used in our
studies because previous experiments had demonstrated that
virtually 100% of animals inoculated with these cells develop
tumors (20). The first group of mice had minimal disease and
was treated shortly after inoculation with NB cells, before
tumors were clinically evident; the second group received treat-
ment once they developed small (,400 mm3) tumors; and the
third group was treated after their tumors were .400 mm3.
After 12 weeks of treatment with TNP-470, 53% of the animals
with minimal disease were tumor free. Furthermore, 14 of these
animals have remained tumor-free after being followed for a
median of 11 months. In contrast, tumors developed in all of the
control mice 3–9 weeks after tumor inoculation. In animals
bearing small tumors, TNP-470 treatment decreased the mean
rate of tumor growth, although tumors continued to increase in
size over time. However, when TNP-470 was administered to
animals with large tumors, the mean tumor growth rate did not
differ significantly from that observed in control animals. Thus,
TNP-470 inhibited tumor growth in animals with minimal disease and decreased the rate of growth in animals bearing small
tumors. This model suggests that there may be a critical NB
tumor burden above which TNP-470 has little efficacy.
Similar to other series, a small number of animals treated
with TNP-470 in our study developed weight loss (12, 21).
Although three animals died from complications related to TNP470 treatment, overall survival for the group of animals whose
treatment was initiated when disease was minimal remained
superior to that of the control group. The TNP-470 dose we used
in our studies was similar to the regimens described by other
investigators in preclinical trials (11, 12, 14). Limited data exist
on the pharmacokinetics of TNP-470 and its known metabolites,
and t1/2 values range from 7 min to 3 h (14). This prompted our
use of a smaller dose given twice daily on treatment days for the
mice with minimal disease. Additional experiments are planned
to evaluate whether treatment with a further reduction in dose
will result in decreased toxicity but similar response rates.
Although TNP-470 is a known inhibitor of endothelial cell
proliferation (22), the mechanisms by which TNP-470 inhibits
tumor growth remain largely unknown. To determine whether
TNP-470 affected NBL-W-N cell growth directly, we performed tumor cell proliferation and cytotoxicity assays. Inhibition of NBL-W-N cell proliferation was not observed until the
concentration of TNP-470 was $10 mg/ml, and cytotoxic effects were not seen until the concentration of TNP-470 was
$100 mg/ml. Because the plasma concentrations of TNP-470 in
animals after s.c. injections have been reported to be ,1 mg/ml
(12), the in vivo antitumor effect observed in our studies does
not appear to be due to decreased NB tumor cell proliferation or
a direct cytotoxic effect.
Histological analyses were performed to compare the tumors from the treated and control groups, and no obvious
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Clinical Cancer Research 4277
Fig. 3 Light microscopy of a
xenotransplanted human NBLW-N NB tumor. H&E staining is
shown. Sheets of neuroblasts
with scant cytoplasm are seen, as
well as typical rosette formation.
Magnification, 3400.
Fig. 4 Proliferation of cells from the MYCN-amplified NB cell line
NBL-W-N. Proliferation was inhibited at TNP-470 concentrations of
$10 mg/ml
morphological differences were observed. Our inability to detect
differences in the degree of apoptosis among the different treatment groups may be explained in part by the high level of
apoptosis that occurs in untreated NB tumors (23, 24). Furthermore, necrosis frequently occurs in large tumors that outgrow
their vascular supply, and the majority of the tumors examined
histologically were .1000 mm3. Surprisingly, we also found
that blood vessel density was not affected by TNP-470 treatment, indicating that some blood vessels can remain intact after
treatment with this agent. Interestingly, Bergers et al. (25) also
reported little change in blood vessel density with angiogenesis
inhibitor treatment in a transgenic model of pancreatic islet cell
carcinogenesis. These authors speculated that the intact vessels
somehow dictate the number of tumor cells that can survive,
thus governing tumor size while maintaining a similar density of
tumor vessels.
The results of this study and the preclinical trials reported
by others (10 –12, 26) demonstrate that the rate of tumor growth
is decreased with TNP-470 treatment. However, TNP-470 appears to have only limited antitumor activity in animals with
clinically evident tumors because tumors do not regress with
TNP-470 therapy, and long-term survival is not affected by
treatment. In contrast, survival was significantly improved in the
group of animals that received TNP-470 treatment 12 h or 1
week after tumor inoculation, indicating that this agent may be
most effective in inhibiting NB growth in the setting of minimal
disease. Our results further suggest that little clinical response
may be observed in Phase I and II studies of TNP-470, in which
most patients have large, bulky tumors. However, TNP-470 may
prove to be effective in children with NB if it is administered
after the completion of intensive multimodality therapy, when
only minimal residual disease is present. In an effort to further
test this hypothesis and to more closely mimic the clinical
scenario of minimal disease, additional studies are ongoing in
which animals with palpable tumors are being treated with
chemotherapy followed sequentially by TNP-470.
ACKNOWLEDGMENTS
We thank Mona Cornwell for technical support in processing the
histological tumor sections and Sharon Lang for performing the terminal
deoxynucleotidyl transferase-mediated nick end labeling assay.
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Effectiveness of the Angiogenesis Inhibitor TNP-470 in
Reducing the Growth of Human Neuroblastoma in Nude Mice
Inversely Correlates with Tumor Burden
Howard M. Katzenstein, Alfred W. Rademaker, Christof Senger, et al.
Clin Cancer Res 1999;5:4273-4278.
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