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www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 13
Combination of metronomic cyclophosphamide and dietary
intervention inhibits neuroblastoma growth in a CD1-nu mouse
model
Raphael Johannes Morscher1,4, Sepideh Aminzadeh-Gohari1, Cornelia Hauser-
Kronberger2, René Günther Feichtinger1, Wolfgang Sperl3, Barbara Koer1
1 Laura Bassi Centre of Expertise-THERAPEP, Department of Pediatrics, Paracelsus Medical University, 5020 Salzburg, Austria
2Department of Pathology, Paracelsus Medical University, 5020 Salzburg, Austria
3Department of Pediatrics, Paracelsus Medical University, 5020 Salzburg, Austria
4 Division of Medical Genetics, Medical University Innsbruck, 6020 Innsbruck, Austria
Correspondence to: Raphael Johannes Morscher, e-mail: raphael.morscher@pmu.ac.at
Keywords: neuroblastoma, ketogenic diet, glucose, metronomic cyclophosphamide, anti-angiogenic
Received: September 15, 2015 Accepted: February 05, 2016 Published: March 05, 2016
ABSTRACT
Background: MYCN-amplication in high-grade Neuroblastoma (NB) tumors
correlates with increased vascularization and therapy resistance. This study combines
an anti-angiogenic approach with targeting NB metabolism for treatment.
Methods and Results: Metronomic cyclophosphamide (MCP) monotherapy
signicantly inhibited NB growth and prolonged host survival. Growth inhibition was
more pronounced in MYCN-amplied xenografts. Immunohistochemical evaluation
of this subtype showed signicant decrease in blood vessel density and intratumoral
hemorrhage accompanied by blood vessel maturation and perivascular brosis.
Up-regulation of VEGFA was not sufcient to compensate for the effects of the
MCP regimen. Reduced Bcl-2 expression and increased caspase-3 cleavage were
evident. In contrast non MYCN-amplied tumors developed resistance, which was
accompanied by Bcl-2-up-regulation. Combining MCP with a ketogenic diet and/or
calorie-restriction signicantly enhanced the anti-tumor effect. Calorie-restricted
ketogenic diet in combination with MCP resulted in tumor regression in all cases.
Conclusions: Our data show efcacy of combining an anti-angiogenic
cyclophosphamide dosing regimen with dietary intervention in a preclinical NB model.
These ndings might open a new front in NB treatment.
INTRODUCTION
Neuroblastoma (NB) represents the most common
extracranial solid childhood cancer. Marked biological
and clinical heterogeneity, pose strong challenges to
optimizing therapeutic interventions for individual
cases. Among others biological risk factors include
MYCN status, tumor histology, cancer cell DNA content
and dened segmental chromosomal aberrations [1–5].
In combination with historical clinical data, tumor biology
allows clinicians to guide therapy by stratifying patients
into internationally accepted risk groups [6, 7]. In low- and
intermediate-risk groups, with overall survival rates above
90%, recent studies have been focusing on reducing
therapeutic toxicity. For patients in the high-risk group,
however, improving treatment efcacy is still central with
overall survival rates close to 50% despite multimodal
therapy protocols [1].
With regard to cellular metabolism NBs share the
characteristic reprogramming to high glucose uptake
recently added to the hallmarks in cancer [8–10]. This
preferential utilization of glucose via aerobic glycolysis
even under sufcient oxygen to shunt pyruvate into
oxidative phosphorylation (OXPHOS) pathway, is
commonly described as Warburg effect [11, 12]. Whereas
defects in single OXPHOS subunits can be a direct
cause of the Warburg effect [13–15], other cancers show
a general pattern of low OXPHOS activity [16–19].
In NB, both primary tumor tissue and xenografts
display generalized low mitochondrial respiratory chain
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activity [20, 21]. This metabolic feature might indicate a
dependency on glucose not only for anabolic processes,
but also for energy production. Based on this theory, we
recently reported a preclinical study showing signicant
tumor growth inhibitory effects of dietary intervention in
a NB xenograft model [21]. In that study we observed that
a mild ketogenic diet (KD) and/or calorie restricted (CR)
diet altered blood metabolic parameters (reduced glucose
and increased beta-hydroxybutyrate levels) and induced
signicant growth inhibition of NB xenografts. Detailed
evaluation of mitochondrial OXPHOS parameters showed
no adaptive response in the NB tumors [21].
These growth inhibitory effects are in line with
preclinical data on cancers of the central nervous system
suggesting that CR-KD might constitute an adjuvant
approach in cancer therapy [22–25]. Effects of CR
and/or KD in diverse preclinical cancer models have
recently been reviewed [26, 27]. Anecdotal case reports
of cancer patients treated with CR-KD support a possible
inhibitory effect on tumor growth, but comprehensive
preclinical and clinical evaluation is lacking [28–30]. Pilot
studies of KD in adult patients showed that it might be
tolerated in patients with advanced cancers [31–33].
Because targeting cancer metabolism is
complementary to current treatment strategies for NB,
it could potentially open a new front against this tumor
entity. The current study therefore aims to investigate
the combination of KD and/or CR diet with a low-dose
metronomic cyclophosphamide (MCP) chemotherapy
regimen. MCP has been shown to effectively inhibit
neoangiogenesis in xenograft models of different
tumor entities [34–36] and is under clinical evaluation
[37–40]. We hypothesize that concurrent targeting of
tumor vascularization and cancer cell metabolism might
constitute a cooperative therapeutic approach.
RESULTS
Inhibition of NB tumor growth by MCP in
combination with dietary intervention
In an effort to exploit a potential synergism of
targeting vascular supply and cancer cell metabolism,
we chose to combine dietary intervention with MCP. In
the presented model, MCP induced signicant growth
inhibition (p < 0.001) of NB xenografts of both cell
lines tested from day 9 onward and extended survival
(p < 0.001) compared to the corresponding standard
diet group without MCP (SD group w/o CTx; Figure 1).
Intriguingly on the metronomic dosing protocol,
growth inhibition of SK-N-BE(2) tumors was more
pronounced versus that of tumors of the reportedly more
chemotherapy sensitive SH-SY5Y cell line [41–43].
In addition to the chemotherapy-induced growth reduction,
SH-SY5Y tumors showed a signicant additional
growth inhibition in all three dietary intervention groups
(Figure 1A1). On day 36, tumor volume in the SD group
was signicantly greater compared to that of the calorie
restricted-standard diet (CR-SD) group (p < 0.05), the KD
group (p < 0.01) and the CR-KD group (p < 0.001). In the
SK-N-BE(2) xenografts tumor growth was signicantly
inhibited in the restricted diet groups (Figure 1B1)
CR-SD (p < 0.05) and CR-KD (p < 0.05). Based on the
strong effect of MCP on SK-N-BE(2) tumor growth,
observed absolute effect size of dietary intervention
was limited. One mouse in the CR-SD group was found
dead on day 22 form an undetermined cause. CR-KD
in combination with MCP resulted in tumor regression
all cases of SH-SY5Y and SK-N-BE(2). The fractional
product of Webb [44] on the last day with available control
tumor data showed synergism for KD and CR-KD for
SH-SY5Y and CR-SD and KD for SK-N-BE(2). For the CR-
SD an additive effect and for the CR-KD in SK-N-BE(2)
antagonism was observed (Supplementary Table S3).
These results need to be interpreted with caution due to
the small tumor size in the SK-N-BE(2) and the lack of
dosing curves [44].
Effect of dietary intervention on blood glucose
and ketone body levels
KD and/or CR caused consistent changes in blood
glucose and ketone body levels in mice carrying the two
different xenograft types. Data are given for day 36 or the
last day of therapy (Figure 2A1–2A2 and 2B1–2B2). Blood
glucose levels were signicantly decreased in all four CR
groups when compared to the SD group. In detail, blood
glucose levels in SD group of SH-SY5Y were signicantly
higher when compared to CR-SD (p < 0.05) and CR-KD
(p < 0.01). Ketone body levels were signicantly elevated
in the CR-KD group (p < 0.001) compared to the SD
group, but not in the CR-SD. Glucose in the SK-N-BE(2)
SD group was signicantly higher compared to CR-SD
(p < 0.01) and CR-KD (p < 0.01). Ketone body levels
were signicantly elevated in both the KD (p < 0.05)
and CR-KD (p < 0.001) groups when compared to the
SD group, but not in the CR-SD group. No signicant
changes in blood glucose were detected for neither of the
KD groups. Detailed blood glucose and ketone body data
for all time points is given in Supplementary Table S1. As
published recently [45] the glucose ketone index (GKI), a
ratio of blood glucose [mM] to blood ketone [mM] levels
might help to monitor therapeutic efcacy in patients on
ketogenic diets. Under the given food regimens, mice
showed a GKI > 12 in the SD groups and GKI < 5 in the
CR-KD diet group, on the last day of therapy. These ratios
are consistent with other preclinical studies in mice [45].
Mean GKI is given in Figure 2A3 and 2B3 and shows
a signicant reduction in all dietary intervention groups
(p < 0.001). GKI for all time points are given in
Supplementary Table S2.
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Tumor and body weight
As tumor volume calculated by the described
formula could be regarded as an approximation of
tumor size, tumor weight was recorded after sacricing
mice. In line with the tumor volumes, tumor weight was
signicantly decreased in all groups on MCP (p < 0.001).
Dietary intervention signicantly enhanced suppression of
tumor growth, evaluated as tumor weight, in all therapy
groups (p < 0.05) except for the KD group of SK-N-
BE(2) (Figure 2A3 and 2B3). No signicant change in the
tumor volume (mm^3) to weight (mg) ratio was observed
(p > 0.05) (Supplementary Figure S1). Treatment with MCP
did not signicantly change mouse weight on the last day
of therapy in mice on SD or KD (p > 0.05) (Supplementary
Figure S2). The combination of MCP with CR caused a
signicant change in mouse weight for all four therapy
groups when compared to the corresponding SD group
(p < 0.05). For one mouse in the CR-KD group therapy
was discontinued due to progressive weight loss on
day 29. Detailed body weight data for all time points is
given in Supplementary Table S1.
Effect of MCP and dietary intervention on
proliferation indices in NB xenografts
To further elucidate the tumor growth inhibitory
effect of the combination of MCP and/or dietary
intervention, we evaluated proliferative activity by
scoring the markers Ki67 and PHH3. The staining patterns
revealed similar effects of MCP and dietary intervention
on proliferation indices in xenografts of the two cell lines.
Ki67 levels did not differ signicantly when comparing the
non-restricted and CR groups receiving MCP treatment to
the corresponding SD group w/o CTx (p > 0.05; Figure 3A1
and 3B1). The expression of PHH3, a marker for M-phase
of the cell cycle, was signicantly lower in the CR
SH-SY5Y xenografts (p < 0.01, Figure 3A2) as well as
in CR SK-N-BE(2) xenografts (p < 0.001, Figure 3B2).
Proliferation indices for each individual subgroup SD/
Figure 1: Dietary intervention enhances the growth inhibitory effect of MCP on NB xenografts. After establishing tumors,
mice were randomized to therapy and control groups as indicated. For xenografts of both cell lines the MCP regimen signicantly inhibited
tumor growth compared to the SD group w/o CTx (p < 0.001). (A1) SH-SY5Y and (B1) SK-N-BE(2) tumor growth curves. Data points
represent mean values ± SEM of the corresponding therapy group (n = 8–12). (A2) and (B2) show Kaplan–Meier survival curves for mice
with SH-SY5Y and SK-N-BE(2) xenografts respectively. Survival was signicantly prolonged in all therapy groups when compared to
the SD group w/o CTx (p < 0.001). The effect of dietary intervention on tumor growth was evaluated by comparing diet groups to the
corresponding SD on MCP. Signicance levels are given for each dietary intervention group compared to SD on MCP and are stacked from
the group with lowest to highest tumor volume. Statistics: ANOVA (p < 0.05) followed by two-tailed Dunnett´s test correcting for multiple
comparisons; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Differences in survival were determined in a univariate analysis with the log-rank test.
Death is coded: tumor volume above 3000 mm3, tumor ulceration or impaired health condition. Abbrev.: SD, standard diet; CR, calorie
restriction; KD, ketogenic diet; w/o CTx, without chemotherapy; MCP, metronomic cyclophosphamide.
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KD and CR-SD/CR-KD are given in Supplementary
Figure S3. Together these results indicate that reduced
cell division might play a role in the growth inhibitory
effect of CR, but does not explain the pronounced effect
of MCP in the more chemotherapy-resistant (SK-N-BE(2))
xenografts.
In vitro 4-hydroperoxycyclophosphamide
(4-HC) sensitivity
Because the in vivo results showed MCP to be more
active against xenografts of the reportedly chemotherapy-
resistant cell line SK-N-BE(2), we compared the in vitro
CP-sensitivity of SH-SY5Y and SK-N-BE(2) to conrm
the data reported in the literature [41–43]. We therefore
titrated 4-HC concentration (active metabolite of CP)
to a dose-range, in which both cell lines showed either
complete cell death (10 nM) or no difference in survival
(0.313 nM) under the conditions tested. At all intermediate
doses SH-SY5Y showed signicantly higher sensitivity
to 4-HC (Supplementary Figure S6). From these data we
hypothesized that, rather than a direct cytotoxic effect,
an indirect effect on xenograft growth may be rendering
SK-N-BE(2) tumors more susceptible to MCP. To our
knowledge this has not been shown in literature before.
Macroscopic observation of intratumoral
hemorrhage
Macroscopic inspection and evaluation of photo
documentation showed a tendency to stronger intratumoral
hemorrhage in the SK-N-BE(2) xenografts compared to
SH-SY5Y xenografts (Figure 4A1 and 4B1). We therefore
quantied macroscopic hemorrhage at day 36 or the last
day of therapy (Figure 4A2 and 4B2). In the SK-N-BE(2)
group on SD w/o CTx, 100% (8/8) of tumors showed
pronounced macroscopic hemorrhage. On therapy with
MCP this was reduced to 20% (5/25), which approximates
the level of hemorrhage observed in SH-SY5Y xenografts
(22%, 2/9) without MCP. In the SH-SY5Y xenografts,
MCP reduced macroscopic hemorrhage to a level of 6%
(2/35). Intratumoral hemorrhage did not vary between
dietary subgroups and is given in Supplementary
Figure S4.
Figure 2: Blood glucose reduction and induction of ketosis goes with reduced tumor weight in mice under MCP.
(2A) SH-SY5Y groups and (2B) SK-N-BE(2) groups. (A1) and (B1) CR signicantly reduced blood glucose levels in mice with both
xenograft types. (A2) and (B2) Ketone body levels (beta-hydroxybutyrate) were consistently elevated in the CR-KD groups and the KD
group of SK-N-BE(2). The trend in the KD group of SH-SY5Y did not reach statistical signicance. (A3) and (B3) Mean Glucose Ketone
Index over the treatment period was signicantly reduced in all dietary intervention groups (p < 0.001). (A4) and (B4) Tumor weight was
signicantly reduced in all groups on MCP when compared to the SD group w/o CTx (p < 0.001; data not shown) and dietary intervention
groups as given. The results are consistent with the tumor volumes calculated in Figure 1A and 1B. Data are shown for day 36 or the last
day of therapy. Mean values ± SEM of the corresponding therapy group are given (n = 8–12). Statistics: ANOVA (p < 0.05) followed by
two-tailed Dunnett´s test correcting for multiple comparisons; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Abbrev.: SD, standard diet; CR, calorie
restriction; KD, ketogenic diet; w/o CTx, without chemotherapy; MCP, metronomic cyclophosphamide.
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Effect of MCP on microscopic tumor
vascularization, hemorrhage and vessel
maturation
To further substantiate the results from macroscopic
inspection, tumor vascularization and intratumoral
hemorrhage were studied on tumor sections stained
with Hematoxylin/Eosin (Figure 5A1 and 5B1). In line
with the macroscopic observations, vascularization and
hemorrhage were signicantly higher in the SK-N-BE(2)
xenografts compared to SH-SY5Y tumors. Upon MCP
treatment, hemorrhage in both xenograft types was
signicantly reduced (p < 0.01). Along with the signicant
reduction of vascularization in the SK-N-BE(2) xenografts
(p < 0.05), maturation of blood vessels was observed in
xenografts of this cell type (Figure 6B). Angiogenesis
scoring across all subgroups is given in Supplementary
Figure S5. The change in vessel structure seemed even
more fundamental than the reduction of vessel number.
Increased nuclear accumulation of HIF1A was evident in
the SK-N-BE(2) xenografts exposed to MCP. This was
accompanied by central regions of cell death (Figure 5C)
and VEGF up-regulation (Figure 5D). Increased VEGFA
could not compensate for the anti-angiogenic effect of
the MCP therapy. It can be speculated that the marked
increase in diffusion distance (as can be seen in Figure 6B1
and 6B2, right) likely contributes to the increasing tissue
hypoxia and cell death as depicted in Figure 5C.
Endothelial cell marker msCD-31 and Van Gieson
staining were used to further characterize the nature of
the prominent hemorrhage in SK-N-BE(2) xenografts.
msCD-31 staining of SK-N-BE(2) tumors showed a poorly
dened vascular pattern largely lacking both mature
vessel structure and a basal membrane. Blood vessels of
all diameters were strongly invaded by NB tumor cells,
resulting in erythrocyte leakage into the tumor (Figure 6B1
and 6B2, left). Only rare blood vessels in the periphery
of SK-N-BE(2) xenografts showed an intact vessel
structure with prominent basal lamina, as was consistently
observed in the SH-SY5Y xenografts (Figure 6A1 and
6A2 left). Under MCP treatment, however, in addition to
a reduction of overall blood vessel density, a pronounced
maturation of blood vessels was evident in SK-N-
BE(2) tumors (Figure 6B1 and 6B2, right). No tumor
cell invasion into vascular structures was observed in
SK-N-BE(2) xenografts exposed to MCP. This change of
pattern was not observed in SH-SY5Y xenografts, where
both treatment groups showed relatively-well developed
vascular patterns (Figure 6A1 and 6A2). Compared to SD
tumors w/o CTx MCP treated tumors showed a tendency
towards decreased cellularity and reduced nucleus/cytosol
ratio in both SH-SY5Y and SK-N-BE(2) xenografts.
Figure 3: Reduced proliferation contributes to the growth inhibitory effect of CR but not MCP. (3A) SH-SY5Y groups
and (3B) SK-N-BE(2) groups. IHC evaluation of proliferation markers on day 36 or the last day of therapy. (A1) and (B1) The fraction
of Ki67 positive stained cells was not signicantly altered by MCP or diet when compared to SD w/o CTx. (A2) and (B2) In xenografts
of both cell lines, PHH3 positive cells per high power eld were signicantly lower in the CR groups. (A3) and (B3) Exemplary tumor
sections stained for PHH3 from mice on SD w/o CTx (left) and CR-KD (right) are shown. Scale bar = 50 µm. Mean values ± SEM of the
therapy group are given (n ≥ 8). Statistics: ANOVA (p < 0.05) followed by two-tailed Dunnett´s test correcting for multiple comparisons;
**p ≤ 0.01; ***p ≤ 0.001. Abbrev.: Ad libitum: SD/KD; restricted: CR-SD/CR-KD. SD, standard diet; CR, calorie restriction; KD, ketogenic
diet; w/o CTx, without chemotherapy. Proliferation indices for individual dietary subgroups are given in Supplementary Figure S3.
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Diametrically opposed regulation of Bcl-2
protein levels in SH-SY5Y and SK-N-BE(2)
tumors under MCP
For both cell lines used in our study, Bcl-2
expression was described previously [46] and was
conrmed by IHC-staining in our study. Under continuous
MCP treatment, SH-SY5Y xenografts of mice on SD
showed a progressive increase in tumor volumes starting
around treatment day 16. Along with this resistance,
signicantly higher Bcl-2 (p < 0.01; Figure 7A)
expression was evident in xenografts exposed to MCP
treatment compared to SD w/o CTx. This is in line with the
literature for NB tumors, which suggest Bcl-2 upregulation
as a mechanism of chemotherapy resistance under
standard treatment [47, 48]. Consistent with the tumor cell
characteristics [48] (chemotherapy resistant and MYCN
amplied) the SK-N-BE(2) cell line showed increased
basal Bcl-2 protein expression levels in the untreated
group when compared to SH-SY5Y. On MCP treatment,
however, Bcl-2 protein levels were signicantly reduced
in the SK-N-BE(2) xenografts (p < 0.001; Figure 7B).
Whereas Bcl-2 staining was more preserved in
vascularized areas, it was particularly low with increased
distance to blood vessels (Figure 7B2, right). Western
blot analysis (Figure 7C) supports the results from IHC.
Whereas SH-SY5Y tumors exhibited slightly increased
Bcl-2 levels upon prolonged MCP treatment, SK-N-BE(2)
tumors showed decreased levels. Concomitant with the
decrease in Bcl-2 levels, an increase in cleaved caspase 3
was observed in MCP-treated SK-N-BE(2) xenografts.
These results support the higher sensitivity of SK-N-BE(2)
tumors to MCP as observed by tumor volume.
DISCUSSION
The present study combines two alternative
therapeutic approaches to reduce NB tumor growth in a
preclinical model. In this setting, administering an anti-
angiogenic MCP regimen with dietary intervention was
able to signicantly inhibit growth of NB xenografts of
distinct genetic background and chemotherapy sensitivity.
In line with our previous work that focused
exclusively on the effect of dietary intervention [21], KD
and/or CR reduced glucose availability and increased
blood ketone body levels in mice. Signicant reductions of
Figure 4: Predominant intratumoral hemorrhage in MYCN-amplied tumors is reduced upon MCP treatment.
(4A) SH-SY5Y and (4B) SK-N-BE(2) tumors. Images of xenograft sites, as well as tumors after explantation are shown. Pronounced
macroscopic hemorrhage in the SK-N-BE(2) SD group w/o CTx compared to all other therapy groups of both cell lines was evident.
(A1) and (B1) Specimens from SD w/o CTx compared to SD on MCP are shown. (A2) and (B2) Relative quantication of macroscopically
visible hemorrhagic tumors w/o CTx (SH-SY5Y n = 9, SK-N-BE(2) n = 8) and groups on MCP (SH-SY5Y n = 35, SK-N-BE(2) n = 25).
Intratumoral hemorrhage did not vary between dietary subgroups and is given in Supplementary Figure S4. Abbrev.: SD, standard diet;
w/o CTx, without chemotherapy; MCP, metronomic cyclophosphamide.
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tumor growth, in addition to MCP treatment, was evident in
all dietary intervention groups except for the SK-N-BE(2)
xenograft group on KD. Although dietary effects in
the SK-N-BE(2) subgroup should be interpreted with
caution due to the small absolute tumor size, results of
different subgroups align with our previous work [21].
This supports the metabolic dependency of NBs and their
limited capacity to adapt to the change in nutrient supply.
In contrast to other studies on the effect of KD and/
or CR on tumor growth [21, 49–51], we not only observed
growth inhibition, but also regression of tumor volume.
Intriguingly, the combination of MCP and CR-KD
induced a signicant reduction of tumor size and complete
growth arrest of NB xenografts of both cell lines tested.
This highlights the importance of combining dietary
intervention with other therapeutic approaches as it
might sensitize tumors to the cytotoxic effects of these
therapeutics. In line with our data, enhancement of anti-
tumor effect has been described in additional preclinical
studies [52–54].
For NB, increased vascularization has been proposed
to correlate with high-risk tumor stage and poor clinical
outcome [55–57]. Targeting neovascularization has
therefore been put forward as a therapeutic option [58–61].
Furthermore, a direct link between MYCN amplication
and enhanced angiogenesis has been described [55, 62, 63].
In accord we observed increased angiogenesis and a
highly invasive growth pattern in xenografts of the
MYCN-amplied cell line. Upon MCP treatment, this
phenotype changed to a mature vessel pattern with strong
perivascular collagen deposition and without vascular
invasion of tumor cells. To our knowledge this effect has
not been described before. The change in vessel structure
presumably resulted in undersupplied tumors due to
the increased diffusion distance. This interpretation is
supported by the increase in hypoxic areas with central
Figure 5: Microscopic evaluation supports an anti-angiogenic effect of MCP and vessel maturation in NB xenografts.
(5A1) SH-SY5Y and (5B1) SK-N-BE(2). Hematoxylin/Eosin staining of NB sections on day 36 or the last day of therapy was scored for
angiogenesis and hemorrhage on a scale from 0-3. In the SK-N-BE(2) xenografts, blood vessel morphology changed to a more mature
pattern as described in detail in Figure 6B1 and 6B2. (C) IHC staining for HIF1A of a SK-N-BE(2) xenograft section from the SD group
w/o CTx (upper) and SD on MCP (lower). Blood vessel (arrow) rarecation and maturation caused a hypoxic pattern in SK-N-BE(2)
xenografts with increased nuclear HIF1A accumulation and areas of marked cell death in the center of hypoxic areas (asterisk). (D) This
correlated to VEGFA up regulation in SK-N-BE(2) xenografts exposed to MCP, as depicted by immunoblotting. Scale bars = 100 µm
(overview) and 50 µm (detail). Mean values ± SEM of the therapy group are given (n ≥ 8). Statistics: unpaired t test (p < 0.05); *p ≤ 0.05;
**p ≤ 0.01; ***p ≤ 0.001. Angiogenesis scoring across all subgroups is given in Supplementary Figure S5. Densitometry for VEGFA levels
is given in Supplementary Figure S8A. Abbrev.: SD, standard diet; w/o CTx, without chemotherapy; MCP, metronomic cyclophosphamide.
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cell death as seen on HIF1A staining. Direct evidence for
nutrient deprivation, however, is pending.
Whereas the SH-SY5Y xenografts in the SD group
developed resistance to the MCP regimen that was
accompanied by up-regulation of Bcl-2 levels, in the
reportedly more chemotherapy-resistant SK-N-BE(2)
xenografts [41–43], sensitivity was retained. The increased
in vitro chemotherapy resistance of the SK-N-BE(2)
cells supports an anti-tumor mechanism of MCP that is
not linked to a direct cytotoxic effect. Previous reports
showed that in vitro hypoxia can trigger an autocrine loop
via increased VEGF expression to induce Bcl-2 mediated
chemotherapy resistance in SK-N-BE(2) cells [64, 65].
Although our model supports ndings of increased
VEGFA expression in response to hypoxia, this loop was
not sufcient to induce Bcl-2-mediated tumor cell survival
in the in vivo setting.
This nding might highlight the importance of a
concomitant decrease in nutrient supply that accompanies
the state of low vascularization in tumors which is often
not prioritized in in vitro studies [64, 65]. Supporting
published work on an anti-angiogenic effect of KD [66],
we observed the strongest reduction of angiogenesis in the
combination of KD with MCP. Whereas both publications
report a rarecation of blood vessels and hemorrhage,
the brous depositions and vessel maturation are only
described in our model.
Figure 6: Evaluation of blood vessels by IHC for msCD-31 endothelial cell marker (A1, B1) and Van Gieson staining
of collagen bers (A2, B2). Images show exemplary sections of (6A) SH-SY5Y and (6B) SK-N-BE(2) xenografts at day 36 or the last
day of therapy. The left upper (overview) and lower (detail) images are taken from tumors w/o CTx, the right upper and lower images are
from tumors exposed to MCP. (A1) and (B1) show IHC staining for the endothelial marker msCD-31. Under both conditions SH-SY5Y
xenografts show well-formed blood vessels, as can be appreciated best in the detail section (A1, lower). In comparison SK-N-BE(2)
xenografts show marked difference in blood vessel morphology between the w/o CTx group and the MCP group (B1). (A2) and (B2)
Van Gieson staining identied the strong perivascular connective tissue in the SK-N-BE(2) group on MCP as collagen deposition (red).
Blood vessels are marked by arrows, Scale bars = 100 µm (overview) and 50 µm (detail). Abbrev.: SD, standard diet; w/o CTx, without
chemotherapy; MCP, metronomic cyclophosphamide.
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Although we are aware of the limitations of
an ectopic xenograft model and potential effects of
additional molecular differences between the cell lines,
the pronounced effect on tumor growth inhibition and
blood vessel maturation could help to further clarify
mechanisms in this molecular subgroup of NBs [62].
It can be hypothesized, that along with the increased
vascularization, also a stronger dependency on vascular
supply is present for promoting tumor growth. Together
with targeting cell metabolism this might constitute an
Achilles heel worth aiming for to improve treatment for
therapy-refractory NB.
Figure 7: SH-SY5Y (A) and SK-N-BE(2) (B) cell lines show divergent regulation of Bcl-2 levels under MCP. (A1) and
(B1) Bcl-2 levels on IHC staining were scored on day 36 or the last day of therapy. In the untreated state, SH-SY5Y xenografts showed
signicantly lower Bcl-2 levels when compared to SK-N-BE(2) xenografts (p < 0.001). On MCP treatment, the two cell lines showed an
opposing response. (A2) and (B2) Exemplary Bcl-2 IHC-stained sections w/o CTx (left) and on MCP (right). (C) On western blot analysis,
along with the reduction in Bcl-2 levels, increased cleaved caspase 3 protein was detected in the SK-N-BE(2) xenografts exposed to MCP.
Densitometry for western blots is given in Supplementary Figure S8B.Scale bar = 100 µm; Mean values ± SEM of the therapy group are
given (n ≥ 8). Statistics: Stundent´s t test (p < 0.05); **p ≤ 0.01, ***p ≤ 0.001. Abbrev.: SD, standard diet; w/o CTx, without chemotherapy;
MCP, metronomic cyclophosphamide.
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MATERIALS AND METHODS
Cell lines
Neuroblastoma xenografts were established with
the cell lines (ATCC, Manassas, VA, USA) SH-SY5Y
(CRL-2266) and SK-N-BE(2) (CRL-2271). SH-SY5Y
is a TP53 wild type, non-MYCN-amplied cell line with
no chromosome 1p loss of heterozygosity and is sensitive
to chemotherapy. The SK-N-BE(2) cell line is highly
resistant to a wide range of chemotherapeutic agents and
is characterized by MYCN-amplication, TP53 mutation
(p.C135F) and chromosome 1p loss of heterozygosity [41].
Cells were cultured in standard conditions as described
earlier [21]. MYCN expression on protein level was
conrmed on western blot and is given in Supplementary
Figure S7.
Animal models and sample preparation
All in vivo experiments were performed in
accordance with protocols approved for this study by the
Salzburg Animal Care and Use Committee (Nr.: 20901-
TVG/44/7-2011). This protocol specically addressed
weight loss observed in response to dietary intervention.
Animals were maintained under specic pathogen-free
conditions and care conformed to the Austrian Act on
Animal Experimentation. Xenograft growth was induced
on the right anks of 5-to 6-week-old female CD-1
nude mice (Charles River, Wilmington, MA, USA) by
subcutaneous injection of a 200 µl suspension of NB
cells (2.7 × 107) in serum-free medium and matrigel
(BD Bioscience, Franklin Lakes, NJ, USA). After
reaching a tumor size of 350 mm3, an oral metronomic
cyclophosphamide treatment was started and mice were
randomized to four dietary therapy groups (SD, CR-
SD, KD and CR-KD; n = 10–12). One control group on
standard diet without chemotherapy was included for both
cell lines (SD w/o CTx; n = 8–9).
Two times per week, tumor volumes were measured
using a caliper and calculated according to the formula
width x height x length / 2. Body weight was recorded and
blood glucose and ketone body (beta-hydroxybutyrate)
levels were monitored using enzyme based methods twice
weekly (Precision Xceed, Abbott Laboratories, North
Chicago, IL). In the ad libitum-fed groups, measurements
were performed after a two-hour fasting period or before
feeding in the calorie restricted groups. Animals in CR
groups were single-housed to control food intake. Ad
libitum-fed mice were group housed. Mice were sacriced
at day 36 or when termination criteria were met (health
status, tumor ulceration or volume of 3000 mm
3
). Images
of the xenografts were recorded at the start of therapy,
and before and after removal of the tumor. Cancer tissue
was snap frozen in liquid nitrogen. One 0.5 cm tumor
slice each was formalin-xed and parafn embedded for
histological analysis.
Food composition and energy content
Mice were fed according to four different regimens:
standard diet ad libitum (SD), calorie restricted standard
diet (CR-SD), long-chain fatty acid-based ketogenic diet
ad libitum (KD) and calorie-restricted ketogenic diet
(CR-KD). Detailed food composition and feeding
protocols to evaluate the dietary intervention on NB-tumor
growth were published previously [21] and are given in the
Supplementary Data (Table S4). In brief, the metabolizable
energy contents were: SD (kcal: fat 9%, protein 33% and
carbohydrates 58%) and mild KD (kcal: fat 78%, protein
14% and carbohydrates 8%) (No. V1535-000 and No.
S9139-E02D; Sniff Spezialdiäten GmbH, Soest, D). Diets
were fortied with vitamins and mineral supplements. For
the CR groups, food intake was restricted to 2/3 of the
respective ad libitum intake, which aligns with clinical
protocols for therapy-resistant epilepsy [67–69].
Metronomic cyclophosphamide (MCP) schedule
CP (Sigma, St Louis, MO, USA) was administered
orally through the drinking water as described previously
[70]. In order to achieve the described oral dose of 40 mg/kg
CP per day, nal CP concentration in drinking water was
0.266 mg/ml [70]. Analogous MCP regimens were applied
to target neoangiogenesis in xenograft models of other
tumor entities [34–36] and have low toxicities [70, 71].
Average water/CP intake per mouse was equal between
different diet groups.
In vitro CP sensitivity
In vitro CP sensitivity of both cell lines was
evaluated by utilizing 4-hydroperoxycyclophosphamide
(4-HC, Niomech, Bielefeld, D), an active metabolite of CP.
Cells were seeded in 96 well plates (25 000/well). After
resting for 48 hours, standard medium was replaced by 100
µl medium containing 4-HC (0.157 – 10 nM). 4-HC was
freshly dissolved in DMSO (Sigma), and control medium
contained the maximum DMSO concentration (0.01%).
After three hours of drug exposure, the medium was
replaced by 100 µl standard medium for 24 hours to allow
the cytotoxicity to fully develop. 3-(4,5-dimethylthiazol-
2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay
was performed following the manufacturer’s instructions
(Life Technologies, Carlsbad, CA, USA). Absorbance
was read at 570 nm with an EnSpire Multimode Plate
Reader (PerkinElmer, Waltham, MA, USA). Results were
normalized to background and are given as metabolic
activity relative to control (100%). The average of two
independent experiments with 6 replicates each is shown.
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(Immuno-)histochemical staining and analysis
Histological staining was performed using
5-µm deparafnized tumor sections of NB xenografts.
Hematoxylin/eosin and Van Gieson staining as well as
immunohistochemistry (IHC) stainings (Bcl-2 (B-cell
lymphoma 2), PHH3 (phospho-histone H3), Ki67) were
carried out within the routine diagnostic setting at the
local pathology department following standard protocols.
Antibodies were used at the following dilutions: 1:100
for anti-Bcl-2 (Dako, Glostrup, Denmark), 1:200 for
anti-PHH3 (Cellmarque, Rocklin, CA, USA) and 1:500 for
anti-Ki67 (Dako). Angiogenesis, intratumoral hemorrhage
and Bcl-2 staining were scored on a scale of 0 (none) to 3
(strong). Proliferation indices were scored by evaluating
the proportion of positively stained nuclei scoring at
least 500 cells per slide (Ki67) or counting the number
of positively stained tumor nuclei per high-power eld
(PHH3/HPF). For adequately sized tumors, at least ve
representative regions were scored; magnication was
400-fold. When the tumor size was too small, stained
nuclei/total tumor cells were counted and converted to a
HPF equivalent. Anti-msCD31 (Abcam, Cambridge, UK)
and anti-HIF1A (hypoxia-inducible factor 1-alpha, Novus,
Littleton, USA) stainings were carried out at a dilution
of 1:50 and 1:100, respectively, following protocols for
IHC-staining described earlier [19, 72]. For detection
EnVision kit reagents (Dako) were used following the
manufacturer’s instructions. Analysis of histological
staining was performed by two blinded investigators, and
the mean of both scores was calculated.
Western blot analysis
Whole-cell lysates for western blot analysis were
prepared using standard radioimmunoprecipitation
assay buffer. In brief, 30 µg protein were separated on
10% acrylamide/bis-acrylamide gels before transfer
to polyvinylidene diuoride membranes (Bio-Rad,
Hercules, CA, USA) using CAPS buffer (10 mM
3-[cyclohexylamino]-1-propane sulfonic acid, pH 11; 10%
methanol). The following primary antibodies were diluted
in 5% w/v BSA (Sigma) in TBS-T: 1:1000 anti-Bcl-2
(Dako), 1:1000 anti-cleaved caspase 3 (Cell Signaling
Technologies, Danvers, MA, USA), 1:30 000 anti-tubulin
(Promega, Madison, WI, USA), 1:1000 anti-VEGFA
(vascular endothelial growth factor, Abcam) and 1:1000
anti-NMYC (Abcam). Horseradish peroxidase-labeled
secondary antibodies were used (Dako) and detection
was carried out with Lumi-Light POD-substrate (Roche,
Basel, Switzerland). Quantication of band intensities was
performed using Image Lab Software 5.2.1 (Bio-Rad) and
adapted to the corresponding loading control.
Statistics
Statistical analysis was performed by Student´s t test
(two groups; p < 0.05) and one-way ANOVA (more than
two groups; p < 0.05). To adapt for multiple comparisons
two-tailed Dunnett´s posttest was used. In these cases
multiplicity adjusted p-value is given. If not mentioned
otherwise, results are given as mean ± SEM. The analyses
were performed using Prism 6 (GraphPad Software, La Jolla,
CA, USA) and SPSS 21 (IBM, Armonk, NY, USA). The
fractional product of Webb was used to determine whether
the observed effects show synergistic or additive nature.
The method uses the formula: i1,2 = i1*i2; where i1,2 is the
inhibitory fraction of the combination of two interventions
i1 (cyclophosphamide) and i2 (diet). When the predicted
response exceeds the measured response, synergism is
claimed (Ratio calculated/hypothetical < 1) [44].
Abbreviations
NB, neuroblastoma; SD, standard diet; CR, calorie
restriction; KD, ketogenic diet; w/o CTx, without
chemotherapy; MCP, metronomic cyclophosphamide.
ACKNOWLEDGMENTS
The authors thank PD Dipl Ing. Dr. J. A. Mayr
for fruitful discussions and M. Prinz und B. Lechner for
excellent technical assistance performing IHC-stainings.
FUNDING
This work was supported by the Vereinigung zur
Förderung der pädiatrischen Forschung und Fortbildung
Salzburg, the Children’s Cancer Foundation Salzburg, the
Paracelsus Medical University Salzburg, single project
grant No.: E-10/12/061-KOF and the Austrian Research
Promotion Agency (822782/THERAPEP). The funders
had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
CONFLICTS OF INTEREST
The authors declare that no competing interests
exist.
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