The brain-penetrating CXCR4 antagonist, PRX177561, increases the antitumor effects of bevacizumab and sunitinib in preclinical models of human glioblastoma
Gravina et al. Journal of Hematology & Oncology
The brain-penetrating CXCR4 antagonist, PRX177561, increases the antitumor effects of bevacizumab and sunitinib in preclinical models of human glioblastoma
Giovanni Luca Gravina 0 2
Andrea Mancini 0 2
Francesco Marampon 0 2
Alessandro Colapietro 0 2
Simona Delle Monache 0 2
Roberta Sferra 0 2
Flora Vitale 0 2
Peter J. Richardson 1
Lee Patient 1
Stephen Burbidge 1
Claudio Festuccia 0 2
0 Department of Biotechnological and Applied Clinical Sciences, Neurobiology Laboratory, University of L'Aquila , Via vetoios snc, Coppito II, L'Aquila , Italy
1 Proximagen Ltd., Babraham Research Campus , Cambridge CB22 3AT , UK
2 Department of Biotechnological and Applied Clinical Sciences, Neurobiology Laboratory, University of L'Aquila , Via vetoios snc, Coppito II, L'Aquila , Italy
Background: Glioblastoma recurrence after treatment with the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab is characterized by a highly infiltrative and malignant behavior that renders surgical excision and chemotherapy ineffective. It has been demonstrated that anti-VEGF/VEGFR therapies control the invasive phenotype and that relapse occurs through the increased activity of CXCR4. We therefore hypothesized that combining bevacizumab or sunitinib with the novel CXCR4 antagonist, PRX177561, would have superior antitumor activity. Methods: The effects of bevacizumab, sunitinib, and PRX177561 were tested alone or in combination in subcutaneous xenografts of U87MG, U251, and T98G cells as well as on intracranial xenografts of luciferase tagged U87MG cells injected in CD1-nu/nu mice. Animals were randomized to receive vehicle, bevacizumab (4 mg/kg iv every 4 days), sunitinib (40 mg/kg po qd), or PRX177561 (50 mg/kg po qd). Results: The in vivo experiments demonstrated that bevacizumab and sunitinib increase the in vivo expression of CXCR4, SDF-1α, and TGFβ1. In addition, we demonstrate that the co-administration of the novel brain-penetrating CXCR4 antagonist, PRX177561, with bevacizumab or sunitinib inhibited tumor growth and reduced the inflammation. The combination of PRX177561 with bevacizumab resulted in a synergistic reduction of tumor growth with an increase of disease-free survival (DSF) and overall survival (OS), whereas the combination of PRX177561 with sunitinib showed a mild additive effect. Conclusions: The CXC4 antagonist PRX177561 may be a valid therapeutic complement to anti-angiogenic therapy, particularly when used in combination with VEGF/VEGFR inhibitors. Therefore, this compound deserves to be considered for future clinical evaluation.
Glioblastoma; CXCR4; Bevacizumab; Sunitinib
Glioblastoma (GBM) is the most common malignant brain
tumor in adults and makes up approximately 5% of brain
tumors in children. Standard of care includes maximal
surgical resection of the tumor, followed by radiation in
combination with chemotherapy. Despite therapeutic advances
over the past decade, the diagnosis of glioblastoma is
associated with a median overall survival time of 15–18 months
and a 5-year survival rate of less than 5% (see review ref
). The failure of standard regimen for GBM can be
accounted for by multiple factors including, but not limited
to, the heterogeneity of the microenvironment, de novo
and/or acquired tumor resistance, and limitations in drug
delivery . Alternative approaches, particularly those that
can target the mechanisms of recurrence, are required.
Therapy failure coupled with the highly vascularized nature
of GBM has led to the consideration of agents targeting
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neo-angiogenesis as alternative therapeutic strategies for
this disease (see review ). Tumor angiogenesis is strongly
regulated by the VEGF/VEGFR system. Bevacizumab, an
anti-vascular endothelial growth factor (VEGF) antibody,
was approved in the USA in 2009 to treat glioblastoma
recurrence on the basis of encouraging preclinical and
clinical results [4–6]. Although there is evidence that
bevacizumab reduces tumor edema, angiogenesis, and disease
burden, the use of this agent as well as other VEGF/
VEGFR-targeting drugs has been followed by resistance
(largely as a result of adaptive tumor responses) in
preclinical models and in clinical settings. Multiple not mutually
exclusive hypotheses attempting to explain this resistance
include direct effects on glioblastoma cells, modifications of
the perivascular niche , increased
bevacizumabmediated hypoxia [13, 14], which increases the triggering of
proliferation activation in cancer stem cells, and the
recruitment of circulating monocytes and macrophages 
which maintains vessel integrity and cancer stem cell
growth. In addition, magnetic resonance imaging (MRI)
performed on patients with bevacizumab-resistant
glioblastoma revealed that these tumors have more diffuse
borders compared to responding tumors. This makes tumor
borders difficult to identify and suggests that resistance to
bevacizumab may be characterized by a more invasive state
[10, 11]. Despite this, data from clinical trials suggest that,
in some patients with recurrent GBM treated with
bevacizumab or tyrosine kinase inhibitors (e.g., sunitinib 
which targets VEGFR, c-kit, and PDGFR), improved
6month progression-free survival rate (PFS) and
radiographic responses [8–11] can be observed.
Macrophages constitute one of the largest cell
populations in glioblastoma [15, 16], and recruitment of myeloid
cells  has been associated with poor responses to
therapy, disease recurrence, and the development of acquired
resistance to therapies, including anti-angiogenic strategies
[15, 16]. It has been demonstrated that Tie2-expressing
monocytes (TEMs), a subpopulation of circulating blood
monocytes, previously identified as pro-angiogenic and
immunosuppressive, are overrepresented at the invasive front
in surgical samples of human glioblastoma and murine
glioma models after anti-VEGF therapy . This condition
leads to an inflammatory and pro-invasive tumor
microenvironment. Recently, it has been reported that inhibition
of angiopoietin 2 overrides the heightened monocyte
invasion induced by anti-angiogenic therapies in gliomas
[16, 17]. In addition, clinical studies indicate that the levels
of circulating cytokines are increased in most cancer
patients who have undergone anti-VEGF therapy.
Recent experimental data suggest that HGF/MET ,
TGFβ/TGFβR , and CXCL12/CXCR4 [20, 21] enhance
the invasive phenotype of GBM after anti-VEGF/VEGFR
therapy and VEGFR inhibitors may up-regulate CXCR4 in
a TGFβR signaling-dependent manner . CXCR4 is a
well-known G-protein-coupled receptor (GPCR) for the
small chemokine stromal-derived factor (SDF)-1α, which
is also known as CXCL12. A second receptor for SDF1α is
CXCR7 which is expressed on vascular endothelial cells, T
cells, dendritic cells, B cells, brain-derived cells, and tumor
cells, including human glioma cells [23, 24]. CXCR4 is
implicated in neo-angiogenesis and vasculogenesis, so we
hypothesized that combining anti-angiogenic therapy with
CXCR4 inhibition would have superior antitumor activity
compared to single treatments. We employed the tyrosine
kinase inhibitor sunitinib and bevacizumab the anti-VEGF
antibody which is selective for human VEGF and shows
low affinity for mouse VEGF. Hence, the efficacy of
bevacizumab in these experiments is due solely to the
sequestration of human tumor cell line-derived VEGF and not
that derived from mouse stromal cells.
Human glioma cell lines U251, U87MG, and T98G were
originally obtained from the American Type Culture
Collection (ATCC, Rockville, MD). A172 and luciferase
transfected U87MG cells were kindly provided by Jari E.
Heikkila (Department of Biochemistry and Pharmacy, Abo
Akademi University, Turku, Finland). Cells were cultured
at 37 °C in 5% CO2 and were maintained in DMEM
containing 10% (v/v) fetal bovine serum, 4 mM glutamine,
100 IU/ml penicillin, 100 μg/ml streptomycin, and 1%
nonessential amino acids (Invitrogen Life Technologies,
Inc., Rockville, MD). To minimize the risk of working with
misidentified and/or contaminated cell lines, the cells used
in studies reported here were stocked at very low passages
and used at <20 subcultures. Periodically, DNA profiling
by GenePrint® 10 System (Promega Corporation, Madison,
WI) was carried out to authenticate cell cultures.
Chemicals and other reagents
All the materials for tissue culture were purchased from
Hyclone (Cramlington, NE, USA). Plasticware was
obtained from Nunc (Roskilde, Denmark). Antibodies for
β-actin [sc-130065], Ang2 (F1) [sc-74403], Ang-1 (C-19)
[sc-6320], MMP-2 [4D3, sc-53630], TGFβ RI (V-22)
[sc398], and CXCR4 [4G10, sc-53534] were purchased from
Santa Cruz (Santa Cruz, CA, USA). Tie2 (AB33, Mouse
mAb #4224) and Phospho-Tie2 (Ser1119, Antibody
#4226) were purchased from Cell Signaling Technology
Europe, B.V. (Leiden, The Nederland). PRX177561 was
provided by Proximagen Ltd. and is a highly selective
CXCR4 antagonist (Ki at human and mouse CXCR4
receptor approximately 3 nM), which shows no activity at
the other chemokine receptors, nor at 75 other drug
targets (enzymes, receptors, and ion channels) at 10 μM.
This compound crosses the blood-brain barrier.
The Matrigel (BD Biosciences, 356237) endothelial
branching morphogenesis assay establishes the potential of ECs to
form tubular networks. For this, 300 μl Matrigel (10.4 mg/
ml, not diluted) was added to the wells of 24-well plates
and allowed to gel at 37 °C for 30 min. Then 40,000
hBMEVC (human brain microvascular endothelial cells,
kindly provided by Philip M. Cummins, School of
Biotechnology, Dublin City University, Ireland, were added to each
well and allowed to invade the material for 6–24 h. The
endothelial cell line was routinely grown in EndoGRO MV
Basal Medium (Millipore, Italia) supplemented with 5%
fetal bovine serum, L-glutamine (10 mM), ascorbic acid
(50 μg/ml), heparin (0.75 U/ml), hydrocortisone (1 μg/ml),
recombinant human epidermal growth factor (5 ng/ml),
EndoGRO-LS Supplement (0.2%), and antibiotics (100 μg/
Cell viability assay
The cytotoxicity of bevacizumab, sunitinib, and/or
PRX177561 was measured by the Cell Counting Kit-8
(CCK-8; Dojindo Molecular Technologies Inc., Tokyo,
Japan). CCK-8 contains Dojindo’s highly water-soluble
tetrazolium salt (WST-8), which produces a water-soluble
formazan dye upon reduction in the presence of an
electron mediator. U87MG, U251MG, and T98G cells were
seeded in 96-well plates at a density of 4 × 103 cells per well
to allow for adhesion overnight. After this, the cells were
treated with different concentration of drugs. After 3 days,
10 μl of the CCK-8 solution was added to each well of the
plate, and the plate was incubated for 3 h in the incubator
(37 °C; 5% CO2). The optical density (OD) of the sample
plate was measured at 450 nm in a microplate reader.
After appropriate treatments, tumor cell cultures, tissue
extracts, and plasma samples were harvested for the
analysis of cytokine and receptor expression. CXCR4
(Cyto Glow CXCR4 [pSer339]), cell-based ELISA, was
purchased from Assay Biotech (Sunnyvale, USA).
Human SDF1α (Quantikine ELISA Kit) were purchased
from R&D systems (Minneapolis, USA). All
determinations were performed in triplicate, according to
manufacturer’s instructions. Data are presented mean ±
standard error (SE). Cytokine levels were normalized to
total protein concentration in tissue lysates.
Mouse glioblastoma xenograft model
Female CD1-nu/nu mice, at 6 weeks of age, were
purchased from Charles River (Milan, Italy) and maintained
under the guidelines established by our Institution
(University of L’Aquila, Medical School and Science and
Technology School Board Regulations, complying with
the Italian government regulation n.116 January 27 1992
for the use of laboratory animals). All mice received
subcutaneous flank injections of 1 × 106 U87MG, U251, and
T98G cells representing models for MGMT negative
(U87MG, U251MG) and MGMT positive (T98G) cells.
Tumor growth was assessed bi-weekly by measuring
tumor diameters with a Vernier caliper (length × width).
Tumor weight was calculated according to the formula:
TW (mg) = tumor volume (mm3) = d2 × D/2, where d
and D are the shortest and longest diameters,
respectively. The effects of the treatments were examined as
previously described . Mice with tumor volumes of
100–150 mm3 were randomized to receive vehicle,
bevacizumab (4 mg/kg iv every 4 days), sunitinib (40 mg/kg
po qd), or PRX177561 (50 mg/kg po qd), or
combinations of bevacizumab and sunitinib with PRX177561.
Vehicle was a mixture of hydroxyl-propyl-β-cyclodextrin
(HPβCD) at 10% in water (pH7) and propylene-glycol
(PG), 25/75 (w/w). Animals were sacrificed by carbon
dioxide inhalation, and tumors were subsequently
removed surgically. A part of the tumor was directly
frozen in liquid nitrogen for protein analysis, and the
other part was fixed in paraformaldehyde overnight for
immunohistochemical analyses. Indirect
immunoperoxidase staining of tumor xenograft samples was performed
on paraffin-embedded tissue sections (4 μm). Briefly,
sections were incubated with primary antibodies
overnight at 4 °C. Next, avidin–biotin assays was done.
Mayer’s hematoxylin was used as nuclear
counterstaining. Tumor microvessels were counted at ×400 in five
arbitrarily selected fields, and the data were presented as
number of CD31+ microvessels/×100 microscopic field
for each group. Ki67 labeling index was determined by
counting 500 cells at ×100 and determining the
percentage of cells staining positively for Ki67. Apoptosis was
measured as the percentage of tunnel positive cells +/−
SD measured on five random fields (×400) on
immunofluorescence (IF) images. The presence of red cells in
tumor tissue and in blood vessels as well as the presence
of microthrombi and bleeding zones was demonstrated
by Martius yellow-brilliant crystal scarlet blue technique.
Tumor hemoglobin levels were also quantified .
Evaluation of treatment response in vivo (subcutaneous
In order to get closer to the parameters used to analyze
the pharmacological efficacy assessments in the man, we
quantified the antitumor effects of different treatments
as previously described [26–29]: (1) tumor volume,
measured throughout the experiment; (2) tumor weight,
measured at the end of experiment; (3) complete
response (CR) defined as the disappearance of the tumor;
(4) partial response (PR) defined as a reduction of
greater than 50% of tumor volume with respect to
baseline; (5) stable disease (SD) defined as a reduction of less
than 50% or an increase of less than 100% of tumor
volume with respect to baseline; (6) tumor progression
(TP) defined as an increase of greater than 50% of tumor
volume with respect to baseline; and (7) time to
progression (TTP). These modalities of analysis reduced both
the differences of single tumor volume measurements in
the time linked to differences of engraftment efficacy of
the tumor cells as well as the individual variability of the
response (even though the mice were inbreed).
Combination index of dual administrations was calculated
accordingly Bruzzese et al. .
Dissociation of U87MG xenografts into single-cell
Tumors were removed in a sterile condition from mice
and washed two to three times with 5–10 ml of PBS/
DMEM basal medium to remove blood and debris.
Tissues were cut into small pieces and minced with a scalpel
blade into tiny pieces to increase the surface area for
trypsinization process. Minced tissues were trypsinized in 3–
5 ml of pre-warmed %0.05 trypsin-EDTA for 10–15 min
at a 37 °C water bath. After digestion, an equal volume of
soybean trypsin inhibitor is added to stop the enzymatic
trypsin reaction. Trypsin inactivation is ensured by
pipetting the suspension up and down several times. Then, the
suspension was pelleted down by centrifuging at 800 rpm
(110 g) for 5 min, and supernatant discarded whereas
tissue pieces were resuspended in 1 ml of sterile DMEM
basal medium. The clumps were dissociated by gently
pipetting up and down (three to seven times) until a smooth
milky single-cell suspension was achieved. The number of
pipetting steps directly depends on the size of particles in
the minced tissue. Lengthy and vigorous mechanical
dissociation should be avoided as it might result in cell death
and a reduction in sphere formation. Next, suspension
was pelleted and washed to remove un-dissociated pieces
and debris: Cell suspension was filtered through a 40-μm
cell strainer into a 50-ml tube. And pelleted cells were
counted and used for new passage in nude mice and for
CXCR4 expression in western blot.
Orthotopic intra-brain model
Following IACUC guidelines in an approved animal-use
protocol, nude mice were inoculated intracerebrally as
follows . Animals were anesthetized with 100 mg/kg
ketamine and 15 mg/kg xylazine. The surgical zone was
swabbed with Betadine solution, and the eyes were coated
with Lacri-lube. The head was fixed in a stereotactic frame
(mouse stereotaxic instrument, Stoelting Europe, Dublin,
Ireland), and a midline scalp incision was made. A small
hole was made at 1.0 mm anterior and 2 mm lateral to the
exposed bregma. A sterile 5-μl Hamilton syringe with a
26-gauge needle was inserted to a depth of 3.0 mm from
the skull surface and withdrawn by 0.5 mm to inject 3 ×
103 U87MG cells in a volume of 3 μl. The injection rate
was set to 1 μl/min. After the implantation of the tumor
cells, the needle was left in place for 5 min to prevent
reflux. The needle was then completely withdrawn from the
brain over the course of 4 min (1.0 mm/min), and the skin
was sutured. Treatments were started 5 days after cell
injection when no luciferase activity was intracranially
detectable. Generally, the first positive mouse images were
obtained from 20 days following intracranial inoculation.
Mice were euthanized when they displayed neurological
signs (e.g., altered gait, tremors/seizures, lethargy) or
weight loss of 20% or greater of presurgical weight. Blood
samples were collected for plasma analysis. All mice were
perfused with PBS; a subset was fixed with 4%
paraformaldehyde. If fixed, the brains were stored in
paraformaldehyde for 24 h, 15% sucrose for 24 h, and then 30% sucrose
for 24 h. The brains were paraffin embedded.
Bioluminescence imaging (BLI) was performed by using
the Alliance Mini HD6 (UVItec Limited, Cambridge,
UK). Briefly, after injection with 150 ug/g D-luciferin
(Synchem UG & Co. Altenburg, Germany) in PBS (i.p.),
animals were anesthetized with 100 mg/kg ketamine and
15 mg/kg xylazine and analyzed for BLI using the
Alliance Mini HD6 machine.
Continuous variables were summarized as mean and
standard deviation (SD) or as median and 95% CI. For
continuous variables not normally distributed, statistical
comparisons between control and treated groups were
established by carrying out the Kruskal-Wallis tests. When
the Kruskal-Wallis test revealed a statistical difference,
pair-wise comparisons were made by
Dwass-SteelChritchlow-Fligner method and the probability of each
presumed “non-difference” was indicated. For continuous
variables normally distributed, statistical comparisons
between control and treated groups were established by
carrying out the ANOVA test or by Student t test for
unpaired data (for two comparisons). When ANOVA test
revealed a statistical difference, pair-wise comparisons were
made by Tukey’s Honestly Significant Difference (HSD)
test and the probability of each presumed “non-difference”
was indicated. Dichotomous variables were summarized by
absolute and/or relative frequencies. For dichotomous
variables, statistical comparisons between control and treated
groups were established by carrying out the exact Fisher’s
test. For multiple comparisons, the level of significance
was corrected by multiplying the P value by the number of
comparisons performed (n) according to Bonferroni
correction. TTP was analyzed by Kaplan-Meier curves and
Gehan’s generalized Wilcoxon test. When more than two
survival curves were compared, the logrank test for trend
was used. This tests the probability that there is a trend in
survival scores across the groups. All tests were two-sided
and were determined by Monte Carlo significance. P values
<0.05 were considered statistically significant. SPSS®
(statistical analysis software package) version 10.0 and StatDirect
(version. 2.3.3., StatDirect Ltd.) were used for statistical
analysis and graphic presentation. We analyzed
KaplanMeier curves [26, 32] in terms of hazard ratios (HRs). This
parameter is an expression of the hazard or chance of
events occurring in the treatment arm as a ratio of the
hazard of the events occurring in the control arm. A hazard
ratio of 2 indicates that treatment of reference is twice
more effective with respect to a control population.
Anti-angiogenic therapies induce the expression of
CXCR4 and SDF1α in experimental glioblastomas
It has been demonstrated that bevacizumab failure and
recurrence show typical malignant behavior in humans
with sarcomatous, spindle cell morphology, mitotic
figures, and necrosis [33, 34]. Bevacizumab failure is also
associated with increased expression and activity of the
CXCR4/SDSF1α pathway . To verify if in vivo
administration of bevacizumab or sunitinib increased
CXCR4/SDSF1α signaling, we treated female nude
micebearing U87MG, U251, and T98G subcutaneous
xenografts with bevacizumab (4 mg/kg iv every 4 days )
or sunitinib (40 mg/kg po qd, ). After 35 days of
treatments, animals were sacrificed and tumor
harvested. Half of the tissues were paraffin embedded while
the other half used for tissue extract preparations and
frozen at −80 °C until use. Immunohistochemical and
ELISA determinations were performed in tissue extracts
and blood samples. In U87MG cells, we find that
bevacizumab and sunitinib reduced tumor weights by about 62
and 42%, respectively (Fig. 1a). Similar percentage
changes were found in U251 (69 and 43%, respectively)
and T98G (68 and 48%, respectively), although there
was a considerable heterogeneity in the size of the
tumors after treatment with bevacizumab and sunitinib,
suggesting variability in the therapy response in different
animals. It is, indeed, possible that larger tumors in the
treated groups were less susceptible to anti-angiogenic
treatment. So we verified if bevacizumab or sunitinib
administration modified the levels of CXCR4, TGFβ, and
ang2 and if this was related to the size of the tumors. As
shown in the western blotting shown in Fig. 1c, no
correlation was found between tumor size and CXCR4 and
expression in untreated tumors whereas treatment with
bevacizumab or sunitinib seemed to cause an increase in
the expression of CXCR4. The statistical analyses of
correlation confirmed this qualitative appearance, indicating
that no correlation was found in untreated tumors
(Fig. 1e) whereas a correlation was observed in treated
animals with bevacizumab and sunitinib (Fig. 1f, g) with
correlation coefficients of 0.9084 (P = 0.0003) and 0.7054
(P = 0.0226), respectively. Bevacizumab (r = 0.8247, P =
0.0054) and sunitinib (r = 0.8954; P = 0.0033) also caused
an increase in TGF-β expression in the larger tumors.
We observed also that Ang 2 expression correlated with
tumor size in the bevacizumab (r = 0.6904; P = 0.0287)
and sunitinib (r = 0.5807; P = 0.0388) treated tumors.
This suggested that high CXCR4, TGF-β1, and Ang2
expression levels might be associated with reduced
sensitivity to these treatments. Increased expression of
CXCR4 and Tie2 was confirmed by
immunohistochemistry in the larger tumors (Fig. 1i). In Fig. 1h, we show
the histological appearance of untreated and treated with
bevacizumab or sunitinib U87 xenografts with resolution
of ×100, ×200, and ×400). We observed that
angiogenesis (confirmed by vessel count or hemoglobin content
in Fig. 2b) was higher in CTRL tumors when compared
to bevacizumab- or sunitinib-treated tumors. In
sunitinib treated tumors, we observed also a significant
accumulation of fibrous stroma as response to increased cell
death especially in the smaller tumor. In parallel, SDF1α
levels were increased by anti-angiogenic therapies but
only in the larger tumors (Fig. 2a). Analyses of
correlation performed in the sunitinib and bevacizumab
treated tumors showed a significant correlation with r =
0.844, P < 0.001 and r = 0.7899, P = 0.0007, respectively,
suggesting that high SDF1α expression was associated
with resistance to angiogenic-based therapy. In order to
determine whether the increased expression of CXCR4
and SDF1α was related to the relative resistance to
antiangiogenic treatment, we analyzed the levels of
hemoglobin as an indirect marker for
increased/decreased angiogenesis. Also in this case, smaller tumors
possessed lower hemoglobin content when compared to
larger tumors (Fig. 2b) and these differences were higher
in treated tumors, with a positive and significant
regression coefficients (r = 0.6945, P = 0.016 and r = 0.7932, P
= 0.0005) respectively in bevacizumab and sunitinib
groups indicating once again a strong correlation
between angiogenesis and sensitivity to therapy.
In addition, GBM xenografts treated with bevacizumab
or sunitinib showed an unusual increase in the production
of VEGF and an elevated expression of HIF-1α (Fig. 2c)
with little difference between smaller or larger tumors.
This is in agreement with that observed with the VEGFR
inhibitor PTK787 (vatalanib) .
Then, we asked ourselves if the differences in tumor
weight could be due to differences in tumor engraftment
efficiency. To address this question, we first evaluated if there
were differences in the randomization of tumors. As stated
in MM, mice were randomized to receive treatments only
when tumor volumes reached 100–150 mm3. The statistical
analyses performed in the different group of tumor,
Fig. 1 (See legend on next page.)
(See figure on previous page.)
Fig. 1 Anti-angiogenic therapies induce the expression of CXCR4 and SDF1α in experimental glioblastomas (1). a Graphical distribution of tumor
weights at the end of treatment cycle of 35 days (mean ± standard deviation, (SD)). Each column included ten tumors. b Size-based grouping of
representative U87-derived tumors from animals of control (three tumors) or treated (three tumors) with sunitinib or bevacizumab. c Western blot
evaluation of single three tissue extracts from smaller and larger tumors in each group of treatment. Each lane was loaded with 100 μg of protein.
CXCR4, TGFβRI, Ang2, and Ang1 expression was normalized versus actin. d Statistical analyses of correlation performed among the levels of CXCR4
(adjusted densitometric units by western blots normalized versus actin) and tumor weight for all 30 tissues (ten tissue extracts for three treatments).
e Statistical analyses of correlation performed on untreated tumors (ten tissue extracts). f Statistical analyses of correlation performed on bevacizumab
treated tumors (ten tissue extracts). g Statistical analyses of correlation performed on sunitinib treated tumors (ten tissue extracts). h, i Histological
appearance (h) and CXCR4 and Tie2 expression (i) in untreated and treated with bevacizumab or sunitinib U87 xenografts with resolution of ×100,
×200, and ×400
Fig. 2 Expression of tumor markers. a Expression of SDF1α levels after treatments and differences with tumor size. Analyses of linear regression performed
in the sunitinib and bevacizumab treated tumors showed a significant correlation in these groups (r = 0.844, P = 0.0002). b Hemoglobin content in treated
and control tumors analyzed according to tumor size. Analyses of linear regression performed in the sunitinib and bevacizumab treated tumors showed r
= 0.69 and P = 0.016. c Western blotting determination of VEGF-A, HIF1α, and GCSF. d Growth curved derived from U87MG cell suspensions obtained from
U87MG xenografts treated for 35 days with bevacizumab and scored as indicated in the text in high CXCR4 expression (cell suspensions from larger
tumors) and low CXCR4 expression (cell suspensions from smaller tumors) and grown for 45 days with or without bevacizumab
demonstrated that randomization was balanced in terms of
initial volumes. No significant difference was, indeed,
observed among the groups [controls (130 mm3 +/− 14,
Mean ± SD), sunitibib (137 mm3 +/− 16), and bevacizumab
(128 mm3 +/− 15)] with respect to tumor volumes at the
start of treatments. In order to investigate in more detail
the relationship between CXCR4 expression and
bevacizumab efficacy, a specific experiment was performed.
U87MG xenografted animals were treated with
bevacizumab (4 mg/kg iv every 4 days for 35 days). At the end of
treatment, tumors were harvested, weighed, and defined as
larger (>338 mg) or smaller (<220 mg) tumors accordingly
with tumor volumes superior or inferior to 75th and 25th
percentiles, respectively. Three out of ten tumors were
considered larger and four out of ten tumors were considered
smaller. These tumors were then digested to obtain a cell
suspension, and the expression of CXCR4 was assessed in
smaller and larger tumors generated after bevacizumab
treatment. As shown in Fig. 2d, smaller tumors expressed
lower CXCR4 levels than larger tumors. For this reason,
ten mice for groups received subcutaneous flank injections
of 2 × 106 cell suspension deriving from smaller and larger
tumors were considered to receive cells with higher or
lower CXCR4 expression levels. When tumor volumes
reached 100–150 mm3, mice were randomized to receive
bevacizumab or vehicle for 45 days. Here, we show that
tumor xenograft derived from cellular suspension of larger
tumors with higher CXCR4 generated under the first
bevacizumab treatment was less responsive to the subsequent
treatment with bevacizumab and grown much more of
tumor originated from smaller tumors with lower CXCR4.
Therefore, this experiment suggests that the amount of
CXCR4 expressed in larger tumors may be involved
reduced efficacy of tumor cells to bevacizumab treatment.
Fig. 3 TTP (time to progression) values for control (CTRL), PRX177561, bevacizumab, and the combination in U87MG tumors (a), U251 tumors (d), and
T98G tumors (g). Kaplan-Meier estimates for rates of progression in U87MG tumors (b), U251 tumors (e), and T98G tumors (h). Statistical analyses for
U87MG tumors (c), U251 tumors (f), and T98G tumors (i). U87MG, U251, and T98G bearing nude mice were treated with bevacizumab (4 mg/kg iv
every 4 days) alone or in combination with or PRX177561 (50 mg/kg po qd) in subcutaneous xenograft models. Tumors were randomized when they
reached a volume of 100–150 mm3 and treated for 35 days. Tumors were measured every 2 days for a total of 17 measurements. After 35 days, animals
were sacrificed and tumors harvested and weighed
In vivo effects of CXCR4 antagonist, PRX177561, alone or
in combination with bevacizumab or sunitinib: TTP
Next, we wanted to assess whether the CXCR4
antagonism can increase the effects of anti-angiogenic
compounds. U87MG, U251, and T98G bearing nude
mice were treated with bevacizumab (4 mg/kg iv
every 4 days) and sunitinib (40 mg/kg po qd) alone
or in combination with or PRX177561 (50 mg/kg po
qd) in subcutaneous xenograft models. Tumors were
randomized when they had reached a volume of 100–
150 mm3 and then treated for 35 days. Tumor sizes
were measured every 2 days. After 35 days, animals
were sacrificed and tumors were harvested and
weighed. Time to progression (TTP) was calculated as
described in . The hazard ratios (HRs) were used
as a parameter to compare treatments . In Figs. 3
and 4, we show the effects observed in U251,
U87MG, and T98G tumors when compared to
bevacizumab (Fig. 3) or sunitinib (Fig. 4). PRX177561 and
bevacizumab were able alone or in combination to
reduce U87MG tumor weight and increase TTP values
(Fig. 3a). The HR values for the rate of progression
were significantly greater for the combination of
bevacizumab with PRX177561 (9.98 compared to control)
than for either agent alone (HRs of 2.70 and 1.96 for
PRX177561 and bevacizumab, respectively, Fig. 3c). A
similar pattern was seen with U251MG and T98G
xenografts with reduced tumor weights and increased
TTP values (Fig. 3). Once again, the tumor
progression HRs for the combinations of PRX177561 with
bevacizumab (HR 10.47 in U251MG tumors and 7.02
in T98G tumors) versus either agent alone was
significantly greater (Fig. 3). It was also noticeable that
Fig. 4 TTP (time to progression) values for control (CTRL), PRX177561, sunitinib (Suni), and the combination in U87MG tumors (a), U251 tumors
(d), and T98G tumors (g). Kaplan-Meier estimates for rates of progression in U87MG tumors (b), U251 tumors (e), and T98G tumors (h). Statistical
analyses for U87MG tumors (c), U251 tumors (f), and T98G tumors (i). U87MG, U251, and T98G bearing nude mice were treated with sunitinib
(40 mg/kg po qd) alone or in combination with or PRX177561 (50 mg/kg po qd) in subcutaneous xenograft models. Tumors were randomized
when they reached a volume of 100–150 mm3 and treated for 35 days. Tumors were measured every 2 days for a total of 17 measurements. After
35 days, animals were sacrificed and tumors were harvested and weighed
the tumor weights after 35 days treatment with the
combination showed very little growth from the initial
100–150 mm3, with a 80–90% reduction in tumor
vessel density and Ki67 proliferation index and a
sizeable increase in apoptosis (Fig. 5).
PRX177561 and sunitinib, alone or in combination,
reduced the tumor weights of all three cell types by
approximately 80%, reduced the tumor blood vessel
density, and increased the TTP (Fig. 4). This was associated
with the modulation of Ki67 proliferation index (Fig. 5a,
b), increased apoptosis (Fig. 5c, d), and increased
palisading necrosis (Fig. 5e, f ). The combination of PRX177561
with sunitinib had significantly greater progression HR
values than with either agent alone in all three cell lines
Fig. 5 a Histological Ki67 staining. Graphical analyses performed in U87MG, U251, and T98G cells. b Ki67 staining. Representative images obtained
from U251 xenografts. Pictures were not counterstained. c TUNEL staining. Graphical analyses performed in U87MG, U251, and T98G cells. d TUNEL
staining. e Palisading necrosis (graphical analyses perfromed in U87MG, U251 and T98G), f Representative images in immunohystochemistry obtained
from U251 xenografts. Representative images in immunofluorescence obtained from U251 xenografts
Fig. 6 (See legend on next page.)
(See figure on previous page.)
Fig. 6 In vivo effects of CXCR4 antagonist, PRX177561, alone or in combination with bevacizumab or sunitinib in orthotopic intra-brain
models: Disease-free survival (DFS) analyses. Luciferase-expressing U87 cells were injected in female nude mice as described, and 5 days
after cell injection, when no bioluminescent lesions were visible, animals were randomly assigned to one of the six different treatment
groups: (1) vehicle (control), (2) PRX177561, (3) bevacizumab, (4) PRX177561 plus bevacizumab, (5) sunitinib, and (6) sunitnib plus
PRX177561. The presence of a bioluminescent signal was considered to define the DFS. After 35 days, treatments were stopped and
animals were followed for the presence of bioluminescent positive lesions in order to calculate the DFS. a DSF graphical representation
in our arms. b Kaplan-Meier rates analyzed for PRX177561 ± bevacizumab. c Kaplan-Meier rates analyzed for PRX177561 ± sunitinib.
d Statistical analysis
(Fig. 4). The combination HR values compared to control
were between 6.73 and 8.35.
In vivo effects of CXCR4 antagonist, PRX177561, alone or
in combination with bevacizumab or sunitinib in
orthotopic intracranial models: DFS and OS parameters
Luciferase-expressing U87 cells were injected in female
nude mice as described and 5 days after cell injection, and
when no bioluminescent lesions were visible, animals were
randomly assigned to one of six different treatment
groups: (1) vehicle (control), (2) PRX177561, (3)
bevacizumab, (4) PRX177561 plus bevacizumab, (5) sunitinib, and
(6) sunitnib plus PRX177561. Since the brain to plasma
ratio of PRX177561 measured at the Cmax value is close
to 1.0, indicating a good permeability through the
bloodbrain barrier, we treated animals bearing orthotopic
intrabrain tumors with the same PRX177561 dose used for
subcutaneous xenografts. The disease-free survival period
(DFS) was defined as the period when no bioluminescent
signal was detectable. After 35 days, treatments were
stopped and animals were followed for overall survival
(OS) determination. Animals were sacrificed when signs
of distress were noticed.
Treatment with bevacizumab, sunitinib, and PRX177561
all increased the DFS period (by 2.2-, 3.1-, and 2.8-fold,
respectively, Fig. 6a). The HR values are shown in Fig. 6d. In
the combination dosing settings, PRX177561 further
increased the DFS period with bevacizumab (by 2.1-fold) and
sunitinib by 1.2-fold. Thus, in the bevacizumab plus
PRX177561 combination, the DFS was 4.7-fold greater than
that seen with the untreated control, and in the sunitinib
plus PRX177561 3.7-fold.
The Kaplan-Meier curves (Fig. 6b, c) demonstrate
the benefit of combining PRX177561 with
bevacizumab and sunitinib.
Consistent with the inhibition of tumor growth, there
was a significant increase in median overall survival (Fig. 7a)
in PRX177561 treated animals from 47 to 71 days (1.5
times) when compared to control (P < 0.005) with a HR =
5.58. Bevacizumab increased OS by approximately 45%
(1.45 times) with a HR = 3.80 when compared to untreated
animals. Similarly, sunitinib increased OS by approximately
77% with a HR = 4.55 when compared to untreated
animals. The addition of PRX-177561 to bevacizumab or
sunitinib further increased median survival to 144 (2.3
times with HR = 3.24) and 107 (1.29 times, HR = 1.88) days,
respectively. The analysis of Kaplan-Meier curves (Fig. 7b,
c) indicated there was a benefit to the combination in the
increase overall survival of PRX177561 both with
bevacizumab (Fig. 7b) and sunitinib (Fig. 7c).
Despite extensive preclinical and clinical research,
glioblastoma remains among the most devastating malignancies.
Because glioblastomas are highly vascular tumors, therapies
that target angiogenesis have generated substantial interest
[3, 4]. The novel CXCR4 antagonist showing good brain
penetration was demonstrated to reduce growth in vitro
and in vivo of GBM cells and induce glioma stem cell
differentiation . So, we investigated the effect of
PRX177561, with and without bevacizumab or sunitinib, by
using subcutaneous and intracranial GBM cell inoculation
in nude mice. Here, we observed that PRX177561 alone
inhibited tumor growth and increased the efficacy of both
bevacizumab and sunitinib resulting in a significant
reduction in tumor growth in animal models of GBM. This
is in agreement with previous experiments using
AMD3100 [22, 38, 41–46] or POL5551 . We show also
that bevacizumab-mediated inhibition of tumor growth can
be amplified by the simultaneous blockade of the CXCR4
and VEGFR. Due to the species selectivity of bevacizumab
(i.e., only the influence of the tumor-derived VEGF would
have been inhibited in these experiments, and the mouse
stromal cell-derived VEGF would have been unaffected),
we also investigated the effect of the small molecule
inhibitor of the VEGFR2 receptor sunitinib in an attempt to
block all VEGFR signaling. Somewhat surprisingly, there
was no significant difference in the ability of bevacizumab
and sunitinib to inhibit the growth of these tumors,
suggesting that at least in these models, stromal cells play little
part in promoting VEGF-mediated neo-angiogenesis of the
growing tumors. However, since CXCR4 blockade also
amplified the effect of sunitinib on the tumor growth, this
provides extra evidence that the SDF-1α/CXCR4 axis plays
a major role in promoting tumor growth. PRX177561 also
enhanced survival in combination with both bevacizumab
and sunitinib in the orthotopic model. Consistent with the
Fig. 7 (See legend on next page.)
(See figure on previous page.)
Fig. 7 In vivo effects of CXCR4 antagonist, PRX177561, alone or in combination with bevacizumab or sunitinib in orthotopic intra-brain models: overall
survival determinations. Luciferase-expressing U87 cells were injected in female nude mice as described, and 5 days after cell injection,
when no bioluminescent lesions were visible, animals were randomly assigned to one of six different treatment groups: (1) vehicle (control), (2)
PRX177561, (3) bevacizumab, (4) PRX177561 plus bevacizumab, (5) sunitinib, and (6) sunitnib plus PRX177561. The presence of a bioluminescent signal
was considered to define the disease-free survival (DFS). After 35 days, treatments were stopped and animals were followed for overall survival (OS)
determination. Animals were sacrificed when a sign of distress was noticed. a DFS graphical representation in our arms. b Kaplan-Meier rates analyzed
for PRX177561 ± bevacizumab. c Kaplan-Meier rates analyzed for PRX177561 ± sunitinib. d Statistical analysis
report of Barone et al. , we observed that the
combination a CXCR4 antagonist with an anti-VEGF antibody was
able to reduce tumor growth and increase overall survival
in intra-brain U87 xenografts. In this study, we showed that
the combination of PRX177561 with bevacizumab, or
sunitinib, increased median TTP values in subcutaneous
xenografts and DFS and OS in intra-brain models when
compared to single treatments. PRX177561 reduced the
expression of Nestin in vivo indicating that CXCR4
antagonism also reduced the maintenance of the cancer
stem cell population as previously demonstrated  It was
interesting to note that the subcutaneous tumors less
susceptible to bevacizumab and sunitinib (i.e., those
larger tumors after treatment) expressed higher levels
of SDF1α, CXCR4, and angiopoietin 2, after blockade
of VEGFR signaling with these two agents. This is
additional evidence these molecules may be associated
with tumor resistance to anti-angiogenic therapy. The
expression of SDF1α and CXCR4 has been strongly
implicated in tumor growth, promoting cell migration
and the recruitment of cells implicated in the
revascularization process in tumors [17, 44–47]; this
strongly suggested that SDF1/CXCR4 assists tumors
in evading anti-angiogenic therapy. The
microenvironment contribution in GBM development is
increasingly emphasized. An interplay exists between CSCs,
differentiated GBM cells, and the microenvironment,
mainly through secreted chemokines causing
recruitment of fibroblasts, endothelial, mesenchymal, and
inflammatory cells to the tumor, via CXCR4. A favorable tumor
microenvironment is also able to increase the growth of
GBM-inducing cancer stem cells to enter in the cell cycle.
Cancer stem cells (CSCs) or tumor-initiating cells (TICs)
drive GBM development, invasiveness, and drug
resistance . It has been demonstrated that CSCs, isolated
from human GBMs, express elevated levels of CXCR4 and
release CXCL12 . Possessing direct antitumor effects,
mainly against cancer stem cell population, PRX177561
associates two fundamental properties to bevacizumab or
Limits of the study
A limit of this study could be that most of the work is
done in a subcutaneous xenograft model, and only a
small part done using an intracranial xenograft model.
However, we have to consider that the subcutaneous
xenograft model, the simplest existing murine model to
study in vivo effects of therapeutic compounds in
GBMs, shows good reproducibility of tumor genesis and
synchronicity of experiments as widely considered in
literature . Subcutaneous tumors typically grow in a
compact form whereas orthotopic tumors display an
infiltrative growth. Therefore, the information obtained
from both models is surely different. Subcutaneous
models offer the opportunity to study in a simple
manner mainly antitumor effects maintaining the opportune
pharmacological selective pressure for the whole
duration of the experiment, i.e., until the time of tumor
sampling. Therefore, subcutaneous model is necessary
for the histochemical, immunohistochemical, and
molecular analyses with the possibility to address the
question whether any phenotypic differences are surely due
to treatments. We used the orthotopic model, instead,
to verify whether the growth of a small amount of cells
injected in the brain, mimicking the tumor recurrence
after previous treatments (i.e., surgery), was influenced
by examined pharmacological treatments. From this
model, we considered two parameters: disease-free
survival (DFS, as the time in which a bioluminescent
positive event was measured for each tumor) and the overall
survival (OS, as time elapsing from the inoculation of
the cancer to euthanasia of animals after a single
treatment cycle which was 35 days). We monitored animals
for longer times (>200 days) when the pharmacological
selective pressure was lost. It is a necessary state that we
did not want to deal the animals for longer times since
chronic treatments could have important side effects as
suggest by clinical data by using different anti-target
therapies (i.e., anti-her2 therapies).
Our study, however, provides evidence for an enhanced
survival effect on GBM-bearing mice which were treated with
combination between PRX177561 and bevacizumab or
sunitinib and represents a significant scientific rationale for
clinical evaluation of this combined therapy that targets
both VEGF/VEGFRs and CXCL12/CXCR4. PRX177561 is
currently being assessed in a phase 1 clinical trial
The authors thank Dr. Simona POMPILI, Human Anatomy laboratory,
Department of Applied Clinical Sciences and Biotechnology, University of
L’Aquila, for having performed tissue slides on which we analyzed the
expression of several markers by immunohistochemistry.
CF and PJR conceptualized and designed the study. AM, FM, AC, SDM, and
RS acquired the data. CF, GLG, FM, and PJR analyze and interpreted the data.
CF and GLG drafted the manuscript. CF, PJR, LP, and SB critically revised the
manuscript. All authors read and approved the final manuscript.
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