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Published OnlineFirst August 5, 2013; DOI: 10.1158/1535-7163.MCT-13-0244
Molecular
Cancer
Therapeutics
Cancer Therapeutics Insights
Chemotherapy Counteracts Metastatic Dissemination
Induced by Antiangiogenic Treatment in Mice
Alessandra Rovida1, Vittoria Castiglioni2, Alessandra Decio1, Valentina Scarlato1,
Eugenio Scanziani2, Raffaella Giavazzi1, and Marta Cesca1
Abstract
The development of resistance and progressive disease after treatment with angiogenesis inhibitors is
becoming a controversial issue. We investigated the experimental conditions that cause multireceptor tyrosine
kinase inhibitors (RTKI) to augment metastasis and whether opportune combinations with chemotherapy could
counteract this effect. The renal Renca-luc tumor was transplanted orthotopically in the kidney of Balb/c mice,
which then were or were not nephrectomized. The Lewis Lung carcinoma (LLC) was transplanted in the tibial
muscle of C57/Bl6 mice. Treatment with the RTKI sunitinib started at different stages of tumor progression,
mimicking neoadjuvant or adjuvant settings. Combination studies with paclitaxel, doxorubicin, cisplatin,
gemcitabine, and topotecan were done on the LLC model, using opportune regimens. In a neoadjuvant setting,
sunitinib inhibited Renca-luc tumor growth, prolonging survival despite an increase in lung metastasis;
treatment after primary tumor surgery (adjuvant setting) or on established metastasis prolonged survival
and decreased metastasis. Sunitinib increased lung metastasis from mice bearing early-stage LLC, but did not
affect established metastases (no acceleration) from advanced tumors. Combinations with doxorubicin,
topotecan, gemcitabine, but not cisplatin and paclitaxel, counteracted the increase in metastasis from LLC,
partly reflecting their antitumor activity. Histology analysis after sunitinib confirmed tumor vascular changes
and increased hypoxia. Topotecan at suboptimal daily doses reduced sunitinib-related metastasis, reducing
tumor hypoxia. Tyrosine kinase inhibitors, as sunitinib, can have adverse malignant effects mainly in the
neoadjuvant setting. The addition of chemotherapy might influence metastasis, depending on each drug
mechanism of action and its regimen of administration. Mol Cancer Ther; 12(10); 2237–47. 2013 AACR.
Introduction
Angiogenesis is required for tumor growth, invasion,
and metastatic dissemination, hence the strong rationale
for an antiangiogenic therapy. Numerous angiogenesistargeting agents have been admitted to the ranks of cancer
therapeutics (1); most of them are used in polytherapy
regimens (2). The most validated antiangiogenic strategy
targets the VEGF axis. VEGF can be blocked directly, as
with the antibody bevacizumab (Avastin), or indirectly by
inhibiting the receptor activity with small molecules such
as multiple tyrosine kinase receptor inhibitors (RTKI).
Authors' Affiliations: 1Laboratory of Biology and Treatment of Metastases, Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche
"Mario Negri"; and 2DIPAV, Faculty of Veterinary Medicine, University of
Milan, Italy
Note: Supplementary data for this article are available at Molecular Cancer
Therapeutics Online (http://mct.aacrjournals.org/).
Corresponding Author: Raffaella Giavazzi, Laboratory of Biology and
Treatment of Metastasis, Department of Oncology, IRCCS - Istituto di
Ricerche Farmacologiche "Mario Negri", Via La Masa 19, 20156 Milan, Italy.
Phone: 3902-3901-4732; Fax: 3902-3901-4734; E-mail:
[email protected]
doi: 10.1158/1535-7163.MCT-13-0244
2013 American Association for Cancer Research.
Among these, sunitinib (Sutent), sorafenib (Nexavar), and
pazopanib (Votrient) have been approved by the U.S.
Food and Drug Administration for a number of malignancies (3). The approval of inhibitors of angiogenesis has
been limited because after an initial prolongation of progression-free survival and improved patient response
rates, they do not always translate into better overall
survival, thus casting doubt on the overall efficacy of
antiangiogenic therapy (4–6). Resistance to antiangiogenic therapy has also been reported both in preclinical
and clinical studies often associated with the activation of
alternative proangiogenic pathways (7, 8).
The progression of a growing tumor to distant metastases involves a number of steps. There is the loss of cell-tocell adhesion, increased motility/invasion, intravasation in
the bloodstream, extravasation, and homing in a different
site. All require permissive angiogenesis, which is also vital
for the survival and proliferation of micrometastases (9–
12). The majority of preclinical studies have focused on the
effect of antiangiogenic therapy on primary tumor growth,
with less attention to metastasis. The results are poor and
controversial. In some studies, antiangiogenic therapy was
extremely effective on the primary tumor and metastasis,
improving survival (13–15). However, surprisingly, recent
studies reported that treatment of tumor-bearing mice,
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mainly with anti-VEGF/VEGFR-related compounds, increased tumor invasiveness and metastasis (16–22). Clinical
data on the effect of antiangiogenesis in general, or antiVEGF therapy, on malignant progression are lacking and
widely debated. With certain tumors, such as glioblastoma,
there was an increase in the volume of infiltrative tumor
after bevacizumab, or a switch to more infiltrating growth
after cediranib (a pan-VEGF-RTKI; refs. 23, 24). A retrospective analysis of five placebo-controlled phase III clinical trials in patients with breast, colorectal, renal, and
pancreatic cancer did not support worse clinical outcome
or altered disease progression after cessation of bevacizumab (25). Results that sunitinib did not accelerate tumor
growth in patients with metastatic renal cell carcinoma
have been recently published (26).
Treatment with bevacizumab generally consists of combinations with standard-of-care chemotherapy, which
might help reduce the unwanted effects of the angiogenesis inhibitors (2). Unlike bevacizumab, the RTKI have
antitumor activity in monotherapy, but there appears to
be no survival benefit from the combination with conventional chemotherapy (27).
In this study, we hypothesized that chemotherapy combined with angiogenesis inhibitors might influence tumor
dissemination and metastasis. We therefore investigated
the effect of the RTKI, sunitinib, alone or with chemotherapy on relevant murine metastatic tumor models. While
sunitinib alone, in the neoadjuvant setting, increased metastasis, the cytotoxic chemotherapy combination counteracted this unwanted tumor dissemination and overcame
the resistance to angiogenesis inhibitors.
Materials and Methods
Animals
Six- to 8-week-old female Balb/c and C57/Bl6 mice
were obtained from Harlan Laboratories and maintained
under specific pathogen-free conditions.
Procedures involving animals and their care were conducted in conformity with institutional guidelines that
comply with National (Legislative Decree 116 of January
27, 1992, Authorization n.19/2008-A issued March 6, 2008,
by the Italian Ministry of Health) and international laws
and policies (EEC Council Directive 86/609, OJ L 358, 1,
December 12, 1987; standards for the Care and Use of
Laboratory Animals, United States National Research
Council, Statement of Compliance A5023-01, October
28, 2008), and in line with Guidelines for the welfare and
use of animals in cancer research (28).
Tumor lines
The renal adenocarcinoma cell line Renca was purchased from American Type Culture Collection (ATCC)
and used within 6 months after receipt; the Renca-Luc
variant was obtained by infecting Renca cells with lentiviral vector carrying the coding sequence of synthetic
firefly luciferase gene, luc2 (Photinus pyralis; ref. 22).
Tumor cells were maintained in RPMI-1640 (Voden Medical Instrument), supplemented with 10% fetal calf serum,
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0.1 mmol/L nonessential amino acids, 1 mmol/L sodium
pyruvate, and 2 mmol/L L-glutamine (Lonza Sales).
Stocks of the cell line were stored frozen in liquid nitrogen
and kept in culture for no more than 3 to 4 weeks before
injection in mice.
The Lewis Lung carcinoma cell line (LLC), obtained
from DTP, DCTD Tumor Repository (www.dtp.nci.nih.
gov), was used within 6 months after receipt, stocked
frozen as in vivo derived tumor fragments and maintained
subcutaneously in C57/BL6 mice for no more than two
generations before testing.
Renca-luc metastatic tumor model
For spontaneous metastasis studies, Renca-luc cell suspensions were inoculated orthotopically (5 105) under the
right renal capsule of BALB/c mice, as previously described (29), and the primary tumor was surgically removed
(nephrectomy) or not. Artificial metastases were obtained
by injecting Renca-luc cells (1 105) into the tail vein (30).
In vivo bioluminescence imaging (BLI) was used to
confirm the presence of tumors, to randomize mice to
start therapy or for nephrectomy, and to follow tumor
growth and metastatic progression (22). Results are plotted as photon count (PC). When specified, ex vivo BLI of
the lung was used to quantify metastatic spread. See
Supplementary Methods for details.
Metastases in the lung were evaluated at the end of
treatment (day 19, interim), or when primary tumors
reached the same size (PC ¼ 2 105, 1 g).
For survival studies, mice were constantly monitored
and killed at the first signs of discomfort (the day of death
being considered the limit of survival). Results are plotted
as the percentage survival against days after tumor transplant. The increment of lifespan (ILS) was calculated as
[(median survival day of treated group median survival
day of control group)/median survival day of control
group] 100.
Lewis lung metastatic tumor model
Tumor cells (5 104) from enzymatically digested subcutaneous tumors were injected into the right tibial muscle
of C57/BL6, as described (30). Primary tumor growth was
measured with a digital caliper three times a week and
tumor volume (mm3) was calculated as: [length (mm) width2 (mm2)]/2.
Animal management and data collection were carried
out with the help of the Study Director 1.8 software
(Studylog System, Inc.). Results were plotted as the mean
tumor volume against days after transplantation. Efficacy
of the treatment was expressed as best tumor growth
inhibition [%T/C ¼ (median weight of treated tumors/
median weight of control tumors) 100].
Animals were euthanized at specific points after treatment started (same day, interim), or when mean primary
tumor volume was 2,000 mm3 (different days, terminal).
Primary tumors were fixed in formalin and embedded
in paraffin (FFPE), or frozen on liquid nitrogen, for further analysis. Lungs were excised and fixed in Bouin’s
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Chemotherapy Counteracts Angiogenic Metastasis
solution (Bio-Optica) and superficial metastatic nodules
were counted and measured using a dissecting microscope, as described in the Supplementary Methods (31).
Drugs and treatments
Sunitinib (Chemietek; 30 mg/kg) and sorafenib (Chemietek; 40 mg/kg) were dissolved in methocell 0.5% and
administered daily orally. Both compounds were used at
or near clinical doses (32). Paclitaxel (Indena S.p.A., 20
mg/kg, i.v.) was dissolved in 50% CremophorEL (SigmaAldrich) and 50% ethanol and further diluted with saline
before use. Cisplatin (Sigma-Aldrich; 5 mg/kg, i.v.) was
dissolved in 0.9% NaCl. Gemcitabine (Eli Lilly and Company; 100 mg/kg, i.p.) was diluted in sterile water. Doxorubicin (Nerviano Medical Science; 6 mg/kg i.v.) was
dissolved in saline. Topotecan (NCI/DTP Repositories,
10 mg/kg, 1 mg/kg or 0.2 mg/kg, i.p.) was dissolved in
PBS. The cytotoxic compounds were administered every
4 days for three cycles (Q4 3), except for 1 and 0.2 mg/kg
topotecan, which were given daily for 10 days.
Histopathological examination
Primary tumors and lungs from euthanized mice were
FFPE for histologic analysis. Micrometastases in the lung
were evaluated by hematoxylin–eosin, as specified in the
Supplementary Methods. Microvessel density and maturation in the primary tumor (CD31 and CD31/a-SMA
double staining), and intratumor hypoxia were evaluated
by immunohistochemistry as described in the Supplementary Methods.
Western blotting
For immunoblotting, total proteins were extracted from
frozen tumor samples. Blots were probed with goat anti
HIF-1a antibody, 1:300 (R&D System) or mouse anti
b-tubulin antibody (1:1000, Sigma Aldrich). See Supplementary Methods for details.
Statistical analyses
Statistical analyses were done using Prism Software
(GraphPad Prism 6 Software). Differences in tumor
growth were evaluated by two-way ANOVA followed
by Bonferroni’s post-test. Differences in survival were
analyzed by the log-rank test. The unpaired Student’s
t test or the nonparametric Mann–Whitney test was used
to compare two groups. For more than two groups, oneway ANOVA followed by Bonferroni’s post-test or the
nonparametric Kruskal–Wallis test followed by Dunn’s
post-test was used. P < 0.05 was considered significant.
Results
Sunitinib in the neoadjuvant setting increases
pulmonary metastases from the renal orthotopic
model Renca-luc
We tested the effect of sunitinib on tumor metastasis in
an orthotopic model of murine renal carcinoma, as the
drug is currently clinical used for this kind of tumor.
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Mice bearing Renca-luc tumors were randomized by
their photon count (PC) to receive either sunitinib or
vehicle (Fig. 1A and B, neoadjuvant setting). Sunitinib
delayed primary tumor growth (Fig. 1A, left) and significantly increased survival (ILS 37%, Fig. 1A, right). Mice
killed at the same primary tumor burden (PC ¼ 2 105,
1 g, Fig. 1B, left), had significantly more lung metastases
with sunitinib than with vehicle (Fig. 1B, right). It is worth
noting that at the end of treatment (day 19, interim), at
a smaller primary tumor by sunitinib treatment did
not correspond less metastases, compared with vehicle
(Supplementary Fig. S1). These findings indicate that the
increase in metastases is not dependent on the effect of
sunitinib on the primary tumor. To determine whether
the sunitinib-related increase also affected established
metastases, in a second experiment, the primary tumor
was surgically removed before treatment (i.e. adjuvant
setting; Fig. 1C). Two days after removal of the primary
tumor, sunitinib treatment started and metastasis progression was followed. As shown in Fig. 1C (left), sunitinib did not influence metastasis progression, and
improved overall survival, though not significantly (ILS
50%, Fig. 1C, right).
To confirm that sunitinib had no adverse effect on
implanted metastases, mice were injected intravenously
with Renca-luc cells and sunitinib treatment started as
soon as lung micrometastases were detectable by BLI (Fig.
1D, left and middle). Sunitinib significantly reduced metastatic growth in the lung, with consequent prolongation
of survival (ILS 19%, Fig. 1D, right). At death, all mice
presented a similar metastatic burden (number of nodules
>300, not shown). Pretreatment with sunitinib for 1 week
before intravenous injection of Renca-luc cells did not
affect metastatic growth in the lung or survival (data not
shown).
The increase in metastases due to sunitinib depends
on tumor stage
We used the murine LLC model, which spontaneously metastasizes to the lung, to confirm the above findings
and to study the effects of sunitinib on metastasis formation at different tumor stages. Mice bearing LLC were
treated with sunitinib, starting when tumors were palpable (Fig. 2A), but no metastasis to the lung was
detectable by histology (Fig. 2A, inset) or when tumors
were more advanced (Fig. 2B) and metastases were
already established (Fig. 2B, inset). When started at an
early stage, the drug had a moderate antitumor effect
(T/C 77%; Fig. 2A, left), but it significantly increased the
number of lung metastases compared with controls
(median 123 and 53, respectively; Fig. 2A, middle),
counted at a comparable primary tumor dimension
(different days, terminal). Sunitinib increased the number of medium and large nodules (diameter > 1 mm),
whereas small metastases (diameter < 1 mm) were not
affected by the treatment. A similar increase was
observed when metastases were evaluated at the end of
treatment (same day, interim) despite sunitinib-treated
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Figure 1. Effect of sunitinib on metastatic Renca-luc tumors. A and B, orthotopic injection, neoadjuvant setting. Renca-luc cells were injected under the renal
capsule (intrakidney, i.k.) in Balb/c mice and on day 5 tumor take was assessed by bioluminescence (BLI) and animals were randomized by photon count
(PC) to treatment (T") with either vehicle (n ¼ 20) or sunitinib (n ¼ 20, 30 mg/kg) for 12 days. Primary tumor growth and metastatic burden were followed
by BLI and expressed as PC. A, effect of sunitinib on the lifespan of mice (10 per group). Primary tumor growth (PC) along the test period (mean SEM,
, P < 0.01; , P < 0.001; left). Effect of sunitinib on survival ( , P < 0.0001; right). B, animals (10 per group) were killed with a median primary
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tumor of 2 10 PC (tumor weight 1 g) on different days (median vehicle 19 (16–26); sunitinib 32 (19–34), and lung metastases were assessed by BLI
(mean SEM). Primary tumor (PC; left); lung metastatic burden (PC; right) at scheduled death (mean SEM; , P < 0.05). C, orthotopic injection, adjuvant
setting. Renca-luc cells were injected i.k. in Balb/c mice, which were nephrectomized (primary tumor resection) on day 6, one day after the BLI
assessment of tumor take and lung metastases implantation. Two days after surgery (day 8), mice (10 per group) were treated (T") with either vehicle
or sunitinib (30 mg/kg) for 12 days; metastatic spread was followed by BLI. Effect of sunitinib on the metastatic spread (PC; left) in mice after surgery
(mean SEM); effect of sunitinib on lifespan (right). D, intravenous injection, artificial metastases. Renca-luc cells were injected into the tail vein (i.v.) of Balb/c
mice, which were treated (T") with either vehicle or sunitinib (30 mg/kg) until scheduled death, starting from day 7 after tumor injection (10 per group). From
left to right, effect of sunitinib on metastatic spread along the test period, followed by BLI (PC; mean SEM; , P < 0.001); one representative mouse of the
vehicle and sunitinib-treated groups imaged on day 17; effect of sunitinib on lifespan ( , P < 0.001).
mice had a smaller primary tumor (Supplementary Fig.
S2A and S2B), again suggesting that the increase in
metastasis was not directly related to the size of primary
tumor.
At a more advanced tumor stage, with established lung
metastases, sunitinib had a similarly modest effect on
primary tumor growth (T/C 72%; Fig. 2B, left), but there
was no significant difference in the number of metastases
(Fig. 2B, middle), with a trend, not significant, in more
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Mol Cancer Ther; 12(10) October 2013
small nodules (Fig. 2B, right). Similar results were obtained
with sorafenib, another RTKI with a similar target receptor
profile (Supplementary Fig. S2).
These findings show for both models that sunitinib
augments metastasis in neoadjuvant-like setting and has
no adverse effect on established metastases, suggesting
that the increase in metastasis is related to the presence of
the primary tumor, and this set the basis for our further
experiments.
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Chemotherapy Counteracts Angiogenic Metastasis
Figure 2. Effect of sunitinib on metastatic LLC. LLC cells were injected in the tibial muscle of C57/BL6 mice, which then received vehicle or sunitinib (30 mg/kg,
3
", 10 per group) for 12 days starting on day 7 (A), when tumors were palpable (60–80 mm ) and there were no lung metastases (A, inset), or from day 11 (B),
when the mean tumor volume was 300 mm3 and lung metastases were established (B, inset). A and B, antitumor activity of sunitinib (mean tumor
volume SEM, P < 0.01, P < 0.001; left); total number of metastatic nodules at death (i.e., primary tumor volume 2,000 mm3; , P < 0.001;
middle); number of metastatic nodules divided per size ( , P < 0.05; , P < 0.01; right).
The combination with chemotherapy curbed the
increase in metastasis driven by sunitinib
Angiogenesis inhibitors are generally used in combination with chemotherapy, which ultimately might limit
or mask the negative effect of these drugs on metastasis
formation.
We investigated this possibility in experiments combining sunitinib and chemotherapy drugs with different
mechanisms of action and efficacy on the LLC model:
paclitaxel, cisplatin, doxorubicin, gemcitabine and topotecan. The antitumor and antimetastatic activities of these
treatments are summarized in Fig. 3 and detailed in
Supplementary Table S1. Sunitinib caused a significant
increase in metastases to the lung, with a modest effect on
the growth of the primary tumor. The effect of the combination followed three patterns: (i) Drugs (i.e., paclitaxel
and cisplatin) which as single agents only minimally
affected metastasis and primary tumor growth (T/C
76% and 83%), in combination with sunitinib moderately
improved antitumor activity but did not counteract the
augmentation of metastasis by sunitinib (Fig. 3A and D);
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(ii) doxorubicin, modestly active on primary tumor
growth (T/C 53%), was significantly active on metastasis
formation; in combination with sunitinib, its antitumor
activity increased and it prevented the sunitinib-driven
metastasis augmentation (Fig. 3B and D); (iii) drugs like
gemcitabine and topotecan, which are highly active on the
primary tumor (T/C 5% and 18%) and on metastasis as
single agents, when combined with sunitinib did not
further increase the antitumor activity, but counteracted
the augmentation of metastasis (Fig. 3C and D).
Sunitinib reduced tumor vasculature, improved
pericyte coverage, and increased intratumor hypoxia
in LLC
To elucidate the vascular alterations within the primary
tumor that may enhance tumor invasiveness (33), microvessel density, area, and maturation were measured in
tumor samples collected after 7 days of treatment with
sunitinib and from time-matched controls (interim). Tumor
vessel density and dimension were significantly lower in
sunitinib than vehicle-treated animals. As expected, the
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Figure 3. Antitumor and antimetastatic activity on LLC of sunitinib in combination with different chemotherapeutic agents. LLC cells were injected in
the tibial muscle of C57/BL6 mice, which were randomized on day 7 (8–10/group, palpable tumors) to receive either vehicle, sunitinib, or one of the
chemotherapeutic agents alone or in combination: A, paclitaxel (PTX, 20 mg/kg) and cisplatin (DDP, 5 mg/kg); B, doxorubicin (Doxo, 6 mg/kg); C, gemcitabine
(Gem, 100 mg/kg) and topotecan (TPT, 10 mg/kg). All drugs were given Q4 3, singly (~) or with sunitinib (Sun, 30 mg/kg, ", daily for 12 days). A to C, antitumor
activity of chemotherapy together with sunitinib (mean tumor volume SEM); D, number of metastatic nodules at scheduled death (i.e. primary tumor
3
volume of 2,000 mm ). For vehicle and sunitinib groups, values in the figure are representative of three independent experiments ( , P < 0.05; , P < 0.01;
, P < 0.001 vs. vehicle, ^^^, P < 0.001 vs. sunitinib). Statistical analysis was done on the corresponding vehicle and sunitinib groups.
percentage of double-positive CD31/a-SMA vessels was
higher in sunitinib-treated tumors, an indicator of mature
vessels (Fig. 4A and C). The reduction of vessel area
induced by sunitinib was accompanied by a significantly
larger percentage of hypoxic areas in tumors treated with
sunitinib than in time-matched controls (Fig. 4B and C).
Metronomic low doses of topotecan counteracted the
metastatic spread elicited by sunitinib, acting on
tumor hypoxia
The findings set out above illustrate the possibility,
reported in several studies, that under certain experimental conditions, angiogenesis inhibitors can promote
a metastatic phenotype, partly by creating an increasingly hypoxic tumor microenvironment. We investigated the effect on metastasis formation of sunitinib with
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topotecan administered daily at suboptimal doses, previously reported to affect the hypoxic tumor microenvironment (34, 35).
The combination of topotecan at the metronomic dose
of 1 mg/kg (TPT-1) with sunitinib gave significantly better
antitumor activity than the single agents alone (T/C 65%,
32%, 3% for sunitinib, TPT-1, and SunþTPT-1) and
reduced the number of metastases, even below those of
untreated mice (Fig 5A–C and Supplementary Table S2).
The decrease in metastases was observed at the terminal
analysis, therefore at a later time, due to the slowing of
the primary tumor growth, once again suggesting that the
effect on metastasis is independent from the size of the
primary tumor and persisted after treatment suspension.
Interestingly, topotecan at the metronomic dose of 0.2
mg/kg (TPT-0.2), had almost no effect on tumor growth
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Chemotherapy Counteracts Angiogenic Metastasis
Figure 4. Pharmacodynamic analyses on LLC from sunitinib-treated and time-matched control tumors. LLC tumor-bearing mice were treated as in Fig. 2A
for 7 days, and vessel morphology was analyzed (5 per group). A, from left to right, vessel number and vessel area, by CD31 immunostaining; vessel
maturation index by CD31/a-SMA double staining (% a-SMA/CD31 double-positive vessels/total vessels; A). B, percentage intratumor hypoxic area
(pimonidazole adduct) per field; (mean SEM; , P < 0.05; , P < 0.01). C, representative images of vessel morphology; from left to right: CD31, a-SMA/CD31
(scale bar. 100 mm), pimonidazole (scale bar. 400 mm).
(Fig. 5B), but combined with sunitinib it reduced metastases too, and significantly counteracted their increase due
to sunitinib (Fig. 5C and Supplementary Table S2).
To examine the mechanism by which topotecan contrasted sunitinib-induced metastatic spread, we analyzed
intratumor hypoxia by pimonidazole staining after metronomic TPT-1 treatment (Fig. 5D, top). TPT-1 alone or
with sunitinib significantly reduced intratumor hypoxia
compared with the sunitinib and vehicle groups. Thus,
hypoxia-induced HIF1-a protein accumulation in sunitinib-treated tumors was lowered by the combination with
TPT-1 (Fig. 5D, bottom). Intratumor hypoxia and HIF1-a
protein levels were not significantly affected in the TPT0.2–treated tumors, probably because of the slight, not
experimentally appreciable, effect of this low dose (data
not shown).
Discussion
The discovery of VEGF as a major regulator of endothelial cell growth and survival paved the way for translating these concepts into clinical practice, by targeting
the VEGF signaling pathway as a way to block angiogenesis and inhibit tumor growth (1). However, the
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overall survival benefits of those antiangiogenic drugs
have been modest: often the response to therapy is short
lasting, and patients become refractory or escape treatment (8, 36). Recent preclinical studies have even shown
increased invasion and metastasis in mouse tumor models treated with certain angiogenesis inhibitors (16–22).
Less clear is whether the resistance/escape to therapy
using angiogenesis inhibitors, with accelerated disease
progression or increased mortality, also happens in
patients. Noteworthy, the preclinical studies were obtained with single-agent treatment, although most clinical
trials with angiogenesis inhibitors are in patients already
treated with- or added to- chemotherapy, which could
well be responsible for the final outcome.
In this study, first we reexamined the experimental
conditions in which small-molecule RTKI (e.g., sunitinib)
boost metastasis, and we found that sunitinib promoted
metastasis only in a neoadjuvant setting. We also confirmed that sunitinib can suppress metastatic growth,
leading to an overall survival benefit for mice. Then, in
the LLC model, we assessed whether chemotherapy could
counteract the proinvasive/prometastatic effects of sunitinib, and found that these effects were chemotherapysensitive and dose schedule–dependent. It is known that
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Figure 5. Antitumor and antimetastatic activity of sunitinib in combination with metronomic topotecan on LLC. LLC cells were injected in the tibial muscle of
C57/BL6 mice, which were treated daily with sunitinib (30 mg/kg, "), starting from day 7 (palpable tumors), alone or in combination with metronomic,
low-dose, topotecan (TPT-1, 1 mg/kg or TPT-0.2, 0.2 mg/kg, ~, 8–10/group), daily for 10 days. A and B, antitumor activity of sunitinib in combination
3
with TPT-1 (A) or TPT-0.2 (B; mean tumor volume SEM); C, number of lung metastases at scheduled death (i.e., primary tumor volume of 2,000 mm ).
, P < 0.001 vs. vehicle; ^, P < 0.05; ^^^, P < 0.001 vs. sunitinib. D, top, percentage intratumor hypoxic area (pimonidazole adduct) per field as from A,
(mean SEM, 5 per group; , P < 0.05; , P < 0.001 vs. vehicle; ^^^, P < 0.001 vs. sunitinib); bottom, Western Blot analysis for HIF-1a, on parallel tissue
samples (2 mice per group).
the host plays a role in the response/resistance to antiangiogenic therapy, but also to chemotherapy (37). Therefore, we chose to work with murine tumor models that
allow us to study metastatic spread in immunocompetent
hosts. Similar results were obtained with immunodeficient nude mice bearing LLC and treated with sunitinib
(data not shown).
When used as a single agent, sunitinib increased spontaneous metastasis to the lung in two murine metastatic
models, the Renca renal carcinoma and the LLC. Increased
invasion and accelerated metastasis with inhibitors of
tumor angiogenesis, including sunitinib, were originally
shown by Ebos and colleagues and Paez-Ribes and colleagues (16, 17). More recent studies have reported that
the adverse effects of angiogenesis inhibitors on metastasis are dose-, drug class-, and tumor model–dependent
(18, 19, 22).
Sunitinib is approved for the treatment of metastatic
renal carcinoma (38). However, we found that in Renca
model transplanted orthotopically in the kidney of mice,
sunitinib, at clinically relevant doses (32) promoted
metastasis in a tumor stage–dependent fashion; it had
negative effects on metastasis dissemination only in the
neoadjuvant setting when metastases were not yet
implanted at the beginning of treatment. Conversely, in
2244
Mol Cancer Ther; 12(10) October 2013
the adjuvant setting, when mice had been nephrectomized and metastases were established, sunitinib did not
exacerbate metastatic progression. This is in contrast with
previous reports of increased metastasis formation after
surgery of the primary tumor (16, 19). However, those
studies used sunitinib at high doses for a short period (a
schedule not reflecting clinical practice), recently reported
to be the only conditions in which metastasis is facilitated
(19, 39).
Interestingly, while leading to an increase in metastasis, sunitinib per se inhibited the growth of the primary
tumor, mirrored by increased mouse survival. Sunitinib
can extend progression free survival and overall survival
in patients with metastatic renal carcinoma (38). Therefore, we believe that in this model and at these experimental conditions, the survival gain is due to sunitinib’s
strong effect on the primary tumor and on established
metastases, hiding the negative influence on metastasis
formation. In light of the above observation, our findings
might explain why an increase in metastasis has never
been observed in the clinical practice and sunitinib therapy is beneficial for patients with metastatic renal cell
carcinoma. In fact, patients do not receive sunitinib in the
neoadjuvant setting, but it is often given later after the
removal of the primary tumor (26, 38).
Molecular Cancer Therapeutics
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Published OnlineFirst August 5, 2013; DOI: 10.1158/1535-7163.MCT-13-0244
Chemotherapy Counteracts Angiogenic Metastasis
Contrary to previous reports in which treatment with
sunitinib before intravenous injection of tumor cells led to
a greater metastatic take (16, 19), in our experimental
conditions, we did not find any conditioning effect to
promote the growth of metastasis. Rather, as recently
reported (39), we can confirm that sunitinib significantly
affects the growth of Renca established lung nodules
(experimental metastasis). Our results are important,
given patients with metastatic RCC continuously treated
with these drugs.
The LLC model enabled us to strengthen the results,
showing that sunitinib (and sorafenib), which per se only
moderately affected primary tumor growth, caused a
dramatic and reproducible increment of lung metastases;
again this was true only when metastases were not yet
implanted at the start of treatment, and not when they
were fully established (late-stage treatment), thus mimicking an adjuvant setting. The increase in metastasis is
evident independently of the day of scheduled death
(different times but same tumor size) or the size of the
tumor (end of treatment, different tumor size).
Sunitinib thus seems to exert its main effect on the initial
escape of metastatic cells from the primary tumor, providing a possible explanation of the discrepancy between
preclinical data (augmentation of metastasis) and clinical
observation (in general no worsen outcome; ref. 26).
Whether augmentation of metastasis was a consequence
of the escape from the sunitinib-treated primary tumor,
one would expect an increased number of micrometastases in the lung. Surprisingly, the distribution of small
metastases was not influenced by the treatment, ruling out
an antiangiogenic effect at the secondary site. The second
question is whether concomitant chemotherapy in some
tumor types and with certain drug indications modifies or
overcomes this aggressive metastatic behavior. The best
combination regimens of antiangiogenic compounds and
conventional chemotherapy or novel biologicals must be
optimized to obtain better therapeutic responses (2). It is
worth noting that no change in disease progression patterns was observed in a retrospective analysis of a number
of clinical trials, with several thousand patients treated
with bevacizumab plus chemotherapy (25).
Having set the appropriate experimental conditions, we
found that an opportune combination therapy can curb
sunitinib’s adverse effects on spontaneous metastasis
from the LLC. However, different cytotoxic drugs affected
metastasis to different degrees, partially reflecting their
intrinsic antitumor activity. Only the combination of
sunitinib with gemcitabine or topotecan (but not paclitaxel and cisplatin) reduced the metastatic burden compared with untreated controls, and there was a strong
effect on the primary tumor as well. This prompts us to
hypothesize that the combination’s final outcome might
be due to the antitumor effect of the chemotherapy. With
doxorubicin, however, there was strong abrogation of the
sunitinib-augmented metastasis, but only a moderate
effect on the primary tumor. Cytotoxic drugs have different activity on the primary tumor and metastatic sites and
www.aacrjournals.org
this was particularly evident for doxorubicin (40, 41).
However, it is very possible that the outcome of chemotherapy is influenced by the tumor microenvironment,
such as vascular cells that can be susceptible to angiogenesis inhibitors in combination with chemotherapy (42).
Furthermore, the host immune system can influence the
response to chemotherapy at multiple levels and ultimately influence metastasis (31, 43). Sunitinib is rarely used in
combination with chemotherapy in clinical practice, as no
clear benefit has been reported combining multitargeted
antiangiogenic RTKI with chemotherapy (27, 44). The
limited advantage we observed combining sunitinib
with chemotherapy raises the question whether these
RTKI differ for example from the anti-VEGF antibody
bevacizumab in their antiangiogenic actions or in their
ability to facilitate chemotherapy drug delivery and activity. Mechanism-based combination therapies aimed at
impeding the "bad" consequences of antiangiogenic therapy and improving the overall therapeutic response are
needed (45).
Several mechanisms have been proposed to explain the
increase in metastasis by angiogenesis inhibitors observed
in preclinical studies. They relate to the acquisition of a
more malignant tumor phenotype, or to changes of the
tumor vasculature, or to abnormal recruitment of proinflammatory cells and production of cytokines (4). Hypoxia is a key mediator of tumor progression, contributing
to poor patient survival (46). After anti-VEGF–based
treatment, tumor hypoxia increases, and HIF-1a is activated, so the tumor responds by adapting or evading it
(46, 47). Pharmacodynamic analyses on sunitinib-treated
LLC did in fact show an impairment of vascularization
with an increase in intratumor hypoxia and upregulation
of HIF-1a. Rapisarda and colleagues reported that topotecan, at low daily doses (metronomic regimen), inhibited
the accumulation of the a subunit of HIF-1 by a mechanism independent of DNA replication-mediated DNA
damage (34), then confirmed in a multi-histology target-driven pilot trial on tumor biopsies from patients
receiving topotecan (48).
We found that (i) metronomic topotecan counteracted
the sunitinib-increased metastasis more than a standard
dose; (ii) even at the suboptimal dose of 0.2 mg/kg, which
does not affect tumor growth, topotecan contained the
increment of metastasis; (iii) metronomic topotecan added
to sunitinib-reduced intratumor hypoxia and inhibited
HIF-1a protein accumulation. Increased activity of bevacizumab in combination with daily low doses of topotecan
was reported to be associated with inhibition of HIF-1a
transcription (35); however, tumor invasiveness was not
evaluated in that study. Although these findings suffer
from the limited specificity of topotecan as an HIF-1a–
targeting drug, we feel the effect of metronomic topotecan
on metastasis is hypoxia-mediated rather than a consequence of a direct effect on tumor cells, because suboptimal
dose of topotecan (0.2 mg/kg) counteracted sunitinibdue increase in metastases without affecting primary
tumor growth. The conclusion that in our model, hypoxia
Mol Cancer Ther; 12(10) October 2013
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Rovida et al.
in the primary tumor is what accelerates metastases, and
appropriate drugs in combination regimens can impair
this, is in line with two recent reports pointed to HIF-1a as a
major mediator of metastasis dissemination induced by
antiangiogenic treatment (20, 21).
Our preclinical findings indicate that the combination of RTKI with chemotherapy can influence metastasis and this might depend on the mechanism of action
of the drug and its regimen. Our study used only two
murine tumors and few angiogenesis inhibitors, so
extrapolation to humans and other angiogenesis inhibitors calls for caution. Recently, different outcomes in
the promotion of metastasis have been shown, depending on the angiogenesis inhibitor, but also on the preclinical tumor model (19).
Angiogenesis therapies targeting VEGF/VEGFR pathways are giving some benefits to patients with cancer.
However, the often modest effects might depend not only
on the lack of response of the tumor, but also on our
inability to use those drugs appropriately. Combining
antiangiogenic compounds with other treatment modalities is a reasonable strategy for the best therapeutic
results. Selecting the right combination and the best
administration regimen could help avoid unwanted
effects and potentiate the final outcome.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: A. Rovida, R. Giavazzi, M. Cesca
Development of methodology: A. Decio, M. Cesca
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): A. Rovida, V. Castiglioni, A. Decio, M. Cesca
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A. Rovida, V. Castiglioni, R. Giavazzi,
M. Cesca
Writing, review, and/or revision of the manuscript: A. Rovida, V. Castiglioni, E. Scanziani, R. Giavazzi, M. Cesca
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): V. Scarlato, E. Scanziani
Study supervision: E. Scanziani, R. Giavazzi, M. Cesca
Acknowledgments
The authors thank J.D. Baggott for editing assistance.
Grant Support
This research was supported by the Italian Association for Cancer
Research (AIRC IG no.10424, and AIRC 5 per mille no.12182; to R.
Giavazzi) and Fondazione CARIPLO (no. 2011-0617; to M. Cesca). A.
Decio is a recipient of a fellowship from the Italian Foundation for Cancer
Research (FIRC).
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.
Received April 1, 2013; revised July 18, 2013; accepted July 27, 2013;
published OnlineFirst August 5, 2013.
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Molecular
Cancer
Therapeutics
Correction
Correction: Chemotherapy Counteracts
Metastatic Dissemination Induced by
Antiangiogenic Treatment in Mice
In this article (Mol Cancer Ther 2013;12:2237–47), which appeared in the
October 2013 issue of Molecular Cancer Therapeutics (1), the authors regret that
in some instances the term "neoadjuvant" is used incorrectly. The term
"neoadjuvant" refers to treatment prior to surgical resection of a primary
tumor. In this case, however, the term "neoadjuvant" is used to describe
experiments where treatment is given to animals with primary tumors, and
then the experiment is terminated.
In the interest of clarification, the authors note that the intent, as specified in
the Discussion section, was to distinguish between treatment in the presence
of a primary tumor and the likely absence of established metastases (mimicking a neoadjuvant setting) from postsurgical treatment without a primary
tumor, but eventually with established metastases (adjuvant). Accordingly,
in some instances they use the descriptions "mimicking neoadjuvant," and a
"neoadjuvant-like setting."
Reference
1. Rovida A, Castiglioni V, Decio A, Scarlato V, Scanziani E, Giavazzi R, et al. Chemotherapy
counteracts metastatic dissemination induced by antiangiogenic treatment in mice. Mol Cancer
Ther 2013;12:2237–47.
Published OnlineFirst December 20, 2013.
doi: 10.1158/1535-7163.MCT-13-0914
Ó2013 American Association for Cancer Research.
270
Mol Cancer Ther; 13(1) January 2014
Published OnlineFirst August 5, 2013; DOI: 10.1158/1535-7163.MCT-13-0244
Chemotherapy Counteracts Metastatic Dissemination Induced by
Antiangiogenic Treatment in Mice
Alessandra Rovida, Vittoria Castiglioni, Alessandra Decio, et al.
Mol Cancer Ther 2013;12:2237-2247. Published OnlineFirst August 5, 2013.
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