Neuropilin-1 Identifies a Subset of Bone Marrow Gr1-Monocytes That Can Induce Tumor Vessel Normalization and Inhibit Tumor Growth

Abstract

Improving tumor perfusion, thus tempering tumor-associated hypoxia, is known to impair cancer progression. Previous work from our laboratory has shown that VEGF-A165 and semaphorin 3A (Sema3A) promote vessel maturation through the recruitment of a population of circulating monocytes expressing the neuropilin-1 (Nrp1) receptor (Nrp1-expressing monocytes; NEM). Here, we define the characteristics of bone marrow NEMs and assess whether these cells might represent an exploitable tool to induce tumor vessel maturation. Gene expression signature and surface marker analysis have indicated that NEMs represent a specific subset of CD11b+ Nrp1+ Gr1- resident monocytes, distinctively recruited by Sema3A. NEMs were found to produce several factors involved in vessel maturation, including PDGFb, TGF-β, thrombospondin-1, and CXCL10; consistently, they were chemoattractive for vascular smooth muscle cells in vitro. When directly injected into growing tumors, NEMs, isolated either from the bone marrow or from Sema3A-expressing muscles, exerted antitumor activity despite having no direct effects on the proliferation of tumor cells. NEM inoculation specifically promoted mural cell coverage of tumor vessels and decreased vascular leakiness. Tumors treated with NEMs were smaller, better perfused and less hypoxic, and had a reduced level of activation of HIF-1α. We conclude that NEMs represent a novel, unique population of myeloid cells that, once inoculated into a tumor, induce tumor vessel normalization and inhibit tumor growth. Cancer Res; 72(24); 1-11. ©2012 AACR.

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Published OnlineFirst December 7, 2012.Cancer Res
Alessandro Carrer, Silvia Moimas, Serena Zacchigna, et al.
and Inhibit Tumor Growth
Monocytes That Can Induce Tumor Vessel Normalization
−Neuropilin-1 Identifies a Subset of Bone Marrow Gr1
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Microenvironment and Immunology
Neuropilin-1 Identifies a Subset of Bone Marrow Gr1
Monocytes That Can Induce Tumor Vessel Normalization
and Inhibit Tumor Growth
Alessandro Carrer
1
, Silvia Moimas
1,2
, Serena Zacchigna
1
, Lucia Pattarini
1
, Lorena Zentilin
1
, Giulia Ruozi
1
,
Miguel Mano
1
, Milena Sinigaglia
1
, Federica Maione
3
, Guido Serini
3
, Enrico Giraudo
3
,
Federico Bussolino
3
, and Mauro Giacca
1,2
Abstract
Improving tumor perfusion, thus tempering tumor-associated hypoxia, is known to impair cancer
progression. Previous work from our laboratory has shown that VEGF-A165 and semaphorin 3A (Sema3A)
promote vessel maturation through the recruitment of a population of circulating monocytes expressing the
neuropilin-1 (Nrp1) receptor (Nrp1-expressing monocytes; NEM). Here, we define the characteristics of bone
marrow NEMs and assess whether these cells might represent an exploitable tool to induce tumor vessel
maturation. Gene expression signature and surface marker analysis have indicated that NEMs represent a
specific subset of CD11bþNrp1þGr1resident monocytes, distinctively recruited by Sema3A. NEMs were
found to produce several factors involved in vessel maturation, including PDGFb, TGF-b, thrombospondin-1,
and CXCL10; consistently, they were chemoattractive for vascular smooth muscle cells in vitro.Whendirectly
injected into growing tumors, NEMs, isolated either from the bone marrow or from Sema3A-expressing
muscles, exerted antitumor activity despite having no direct effects on the proliferation of tumor cells. NEM
inoculation specifically promoted mural cell coverage of tumor vessels and decreased vascular leakiness.
TumorstreatedwithNEMsweresmaller,betterperfusedandlesshypoxic,andhadareducedlevelof
activation of HIF-1a. We conclude that NEMs represent a novel, unique population of myeloid cells that, once
inoculated into a tumor, induce tumor vessel normalization and inhibit tumor growth. Cancer Res; 72(24); 1–
11. 2012 AACR.
Introduction
Over the last several years, multiple evidences have indicat-
ed that different bone marrow–derived myeloid cells partici-
pate in various aspects of cancer development and progression
(1–3). Among these are tumor-associated macrophages (TAM),
which exert proangiogenic and protumorigenic activity (4).
Other leukocyte populations recruited at the site of tumor
growth are Tie-2 expressing monocytes (TEM; ref. 5) and
myeloid-derived suppressor cells, a variegated group of
CD11bþGr1þcells able to foster tumor progression and
angiogenesis (6). Finally, tumor-derived TGF-bcan drive the
polarization of neutrophils to acquire a protumoral phenotype
(7). Of note, virtually all of the bone marrow–derived cell types
that infiltrate human tumors appear to possess proangiogenic
activity (1) and their ablation is detrimental to tumor growth
(8), a concept that is also currently being evaluated in clinical
trials (9).
In growing tumors, leukocytes contribute to the excessive
production of proangiogenic factors, which leads to the for-
mation of an abnormal vasculature, typically characterized by
elevated vascular density, abnormal vessel morphology, and
excessive leakiness (10–12). These features seem to influence
tumor progression positively, by inducing a constant state of
hypoxia and acidosis, which, in turn, favors the production of
tumorigenic trophic factors, the natural selection of malignant
cells and the further recruitment of protumorigenic myeloid
cells (13). "Normalizing" the tumor vasculature might thus
represent an effective strategy in cancer therapy, by exerting a
negative effect on malignant progression, blunting tumor
hypoxia, reducing vascular permeability, and enhancing drug
penetration (14, 15).
Here, we describe a novel myeloid cell population that,
when injected into growing tumors, exerts antitumoral
activity by inducing tumor vessel maturation. In recent
years, we and others have observed that the overexpression
Authors' Affiliations:
1
Molecular Medicine Laboratory, International Cen-
tre for Genetic Engineering and Biotechnology (ICGEB);
2
Department of
Medical Sciences, Faculty of Medicine, University of Trieste, Trieste; and
3
Department of Oncological Sciences and Division of Vascular Biology,
Institute for Cancer Research and Treatment (IRCC), University of Torino
School of Medicine, Candiolo, Italy
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/)
A. Carrer, S. Moimas, and S. Zacchigna contributed equally to this work.
Corresponding Author: Mauro Giacca, ICGEB, Padriciano 99, 34149
Trieste, Italy. Phone: 39-040-375-7324; Fax: 39-040–375-7380; E-mail:
giacca@icgeb.org
doi: 10.1158/0008-5472.CAN-12-0762
2012 American Association for Cancer Research.
Cancer
Research
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of the 165 amino acid isoform of VEGF-A (VEGF-A
165
)in
adult tissues determines the recruitment of cells positive for
the myeloid antigen CD11b (16–18) and that these express
the neuropilin-1 (Nrp1) receptor (17). These cells turned out
to be essential in mediating muralcellcoverageofthe
vasculature formed upon VEGF stimulation. Of note, sema-
phorin-3A (Sema3A), which also binds Nrp1 similar to
VEGF-A
165
, also stimulated massive infiltration of these
CD11bþcells in the expressing tissues (17). These, together
with other results, clearly pointed out that Nrp1 was an
essential receptor to mediate the recruitment of these cells.
The infiltrating cells, which originate from the bone marrow
and were negative for lineage-specific leukocyte antigens,
among which CD3, CD4, CD8, and CD49b, are herein referred
to as neuropilin-1 expressing monocytes (NEM).
Here, we characterize NEMs as a population of CD11bþ
Nrp1þGr1cells, which constitutes approximately 1.0% of the
total number of bone marrow cells. We report that the intra-
tumoral injection of NEMs, purified from either the bone
marrow or Sema3A-expressing muscles, is capable of markedly
reducing tumor growth by promoting the formation of a more
mature vascular network, eventually resulting in enhanced
tumor perfusion.
Materials and Methods
Animal studies
C57BL/6 or Balb/c mice were injected subcutaneously
(1 10
6
cells/mouse) with B16.F10 melanoma [CRL-6475;
American Type Culture Collection (ATCC)] or 4T1 breast
carcinoma cells (CRL-2539; ATCC), respectively. Tumor vol-
ume was evaluated daily using calipers and expressed in mm
3
using the formula: V¼p/6 (d
max2
d
min
/2; ref. 19).
Flow cytometry and cell sorting
Bone marrow pools from 4 animals were used for flow
cytometry using the following antibodies: anti-CD45-PE,
anti-CD31-PE, anti-Flk1-PE, anti-Sca-1-PE, anti-c-kit-PE,
anti-CD11c-PE, anti-CD14-PE, anti-isotype controls (all from
Pharmingen Becton Dickinson), anti-CCR2-PE, anti-Flt-1-PE
(from R&D), anti-CXCR4-PE, anti-CD49b, anti-F4/80-PE, anti-
MHCII-PE (from eBioscience), anti-CD11b AlexaFluor647,
anti-Tie-2-PE, anti-B220-PE (all from Biolegend), anti-Ly6C-
PE, anti-CD25-PE, anti-CD16/32-PE, anti-Gr1-perCP (from
Miltenyi Biotec), and anti-Nrp1 (C-19; Santa Cruz Biotechnol-
ogy). Sorting was carried out on a FACSAria II Instrument
(Becton Dickinson Biosciences).
In vivo cell tracking
Sorted NEMs or Gr1þcells were labeled with the fluorescent
dye DiD (Invitrogen) following the manufacturer's instruc-
tions. Cells were subsequently injected into B16.F10 tumors
and animals were sacrificed 6 days after injection.
Migration assays
Migration assays were conducted using 5-mm pore size
Transwell permeable supports (Costar, Corning Incorporated).
Cells (2.5 10
5
) were seeded in serum-free medium in the
upper chamber, whereas recombinant Sema3A (500 ng/mL;
R&D) or FBS (10%) was placed in the lower chamber. After
overnight incubation, migrated cells on the lower chamber
were labeled with Hoechst (2 mg/mL, Invitrogen). Each assay
was carried out in triplicate.
Tissue analysis
Tissues were harvested as described (17). The following
primary antibodies were used diluted 1:200 in blocking buffer:
anti-CD11b, anti-Gr1, anti-CD31 (all from Pharmingen Becton
Dickinson), anti-Nrp1 (R&D Systems), anti–proliferating cell
nuclear antigen (PCNA; F-2; Santa Cruz Biotechnology), Cy3-
coniugated anti-a-SMA (1A4; Sigma), and anti-NG2 (Chemi-
con). AlexaFluor-conjugated antibodies (1:1,000; Molecular
Probes) were used as secondary antibodies.
For hypoxia detection, mice were injected intraperitoneally
with 60 mg/kg of Hypoxyprobe-1 (HPI Inc.). After 1 hour,
animals were sacrificed and tumor masses were harvested
and processed. Tissue slices were fixed with PFA 4% for 30
minutes at room temperature; unmasking with citrate buffer
was carried out for 20 minutes at 98C and slices were then
incubated with the anti-Hypoxyprobe-1 fluorescein isothiocy-
anate–conjugated antibody (HPI Inc.) for 40 minutes in the
dark.
Fluorescence signals were quantified counting the number
of pixels that exceeded a fixed background threshold.
In vitro proliferation assay
B16.F10 cells (1 10
5
) were labeled with calcein (500 ng/mL
for 30 minutes at 37C; Invitrogen), seeded onto a 24-well
plate, and counted after 6 hours; then, freshly purified NEMs
or Gr1þcells were added. After an additional 40 hours, cells
were incubated with Hoechst dye (2 mg/mL; Invitrogen)
for 15 minutes and counted. The proliferation index
was calculated as a ratio between cell count at t¼40 and
t¼0hours.
Vessel permeability assay
Vessel permeability was analyzed by an adaptation of the
Miles' assay. Mice were injected intravenously (i.v.) with 250 mL
0.5% Evans blue (Sigma) and sacrificed after 30 minutes. The
tumor mass and liver were removed and weighted. The dye was
extracted by incubation in 2% formamide at 55C and spec-
trophotometrically quantified at 610 nm. For each animal,
Evans blue content was expressed as a ratio between tumor
and liver content.
Doxorubicin quantification
Doxorubicin (Sigma) was injected i.v. (10 mg/kg) in tumor-
bearing C57BL/6 mice 4 hours before sacrifice. Tumors and the
liver right lobe were harvested and weighted. Tissue-bound
doxorubicin was assessed as described previously (20), nor-
malized according to tissue weight, and expressed as a ratio
between tumor and liver content.
Power Doppler imaging
Power Doppler images from subcutaneously growing tu-
mors (n¼5 per group) were obtained with a Vevo 770 system,
using a 30 MHz probe. Power Doppler signals were assessed at
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3 points along the longitudinal axis, located 0.2-cm away from
each other.
Recovery of NEMs from Sema3A-expressing muscles
Production and use of the recombinant AAV2-Sema3A
(rAAV2-Sema3A) vector was as described previously (17).
After 15 days, mice injected in the tibialis anterior of both
legs [5 10
10
viral particles (vp) per leg] were sacrificed, and
muscles were removed and mechanically minced. Tissue was
digested with collagenase (0.225%; Worthington) at 37C for
60 minutes and filtered. The muscle-derived NEMs (mNEM)
were purified using anti-CD11b–conjugated magnetic beads
(Miltenyi Biotec).
Proliferation and migration of vascular smooth muscle
cells
Vascular SMCs (vSMC; 1 10
5
) from coronary artery
(Clonetics, Cambrex) were seeded in a 24-well plate. After
6 hours, cells were incubated with EdU (Invitrogen) for 4
hours. Then, cells were washed, the medium was replaced,
and freshly purified NEMs or Gr1þcells (2 10
4
)were
added. Twenty hours after EdU addition, cells were washed
twice in PBS, fixed in PFA 4%, and stained with a-SMA
antibody (Sigma) for 2 hours at room temperature (1:400).
EdU detection was carried out using Click-iT EdU Cell
Proliferation Assay (Invitrogen).
Migration assays were conducted using 8-mm pore size
Transwell permeable supports (Costar, Corning Inc.). vSMCs
were stained with calcein (Invitrogen). Then, 1 10
5
cells were
seeded in the upper chamber, whereas purified NEMs or Gr1þ
cells or FBS (20%) were added to the lower chamber. After
overnight incubation, migrated cells were labeled with Hoechst
(2 mg/mL) and double-positive cells were counted. Assays were
carried out in triplicate.
Real-time PCR amplification
Total RNA was extracted using TRIzol reagent (Invitrogen)
and reverse transcribed using hexameric random primers
(Invitrogen). PCR conditions are reported in Supplementary
Table S1.
Statistical analysis
Data were analyzed using a 2-tailed Student t-test or a
nonparametric Mann–Whitney Utest, with P<0.05 considered
significant. Growth curves were analyzed by 2-way ANOVA for
repeated measures and post-hoc analysis. Cluster analysis was
conducted using the Genesis software (21). Data were inter-
nally normalized according to the mean centering method.
Results
Characterization of NEMs from the bone marrow
A freshly isolated bone marrow pool (n¼4) was stained
for CD11b, Gr1, and Nrp1 and analyzed by flow cytometry
and cell sorting. The monocytic fraction was discriminated
according to morphological gating and CD11b positivity
(Fig. 1A, a and b). According to the expression of the Gr1
antigen, mouse monocytes are commonly divided into
Gr1(resident) or Gr1þ(inflammatory) cells (22–24); this
antigen was thus used to sort apart the 2 cell populations
(Fig. 1A, c). Next, both Gr1þand Gr1cells were further
sorted according to expression of Nrp1 (Fig. 1A, d and e). On
the basis of the combined expression of Nrp1 and Gr1, 4
subsets of bone marrow–monocytic cells were thus eventu-
ally obtained (Fig. 1A, f).
Our previous findings indicated that the cells attracted in
vivo by Sema3A were positive for Nrp1 and negative for Gr1
(17). Therefore, we definedbonemarrowNEMsasthe
Nrp1þGr1subset of CD11bþcells, which corresponded
to approximately 1% of total bone marrow cells (in green
in Fig. 1A, f).
Next, we wanted to verify the response of the 4 sorted
populations to Sema3A. Cells were seeded onto the upper
chamber in a Transwell plate and migration in response to
rSema3A was evaluated after 12 hours. Sema3A chemoat-
tracted only the Gr1subset of the Nrp1þcells (Fig. 1B).
We then determined the gene expression profiles of the 4
sorted CD11bþcell subsets. In particular, we assessed the
levels of mRNAs of growth factors and receptors known to be
involved in blood vessel formation, and followed this with
multivariate analysis to detect similarities. A graphic repre-
sentation of the relative gene expression levels in the 4 cell
subsets is shown in Fig. 1C, along with the results of hierar-
chical clustering linkage (HCL). This analysis indicated that the
2 Nrp1þcell populations were more closely clustered accord-
ing to the gene panel analyzed. At the same time, this gene
expression analysis also indicated that the 4 cell subsets
possessed distinct expression signatures.
Comparative flow cytometric characterization of NEMs
and of the other purified cell populations according to
various cell surface antigens is shown in Fig. 1D. The CCR2
antigen showed a different pattern of expression in the 4 cell
subsets. Most of the Gr1þcells expressed this receptor,
however at relatively low levels; in contrast, expression was
more variable in the Gr1subset (NEMs). The CD11c
adhesion molecule was expressed at low levels, in both the
Gr1þand the Nrp1Gr1subsets, whereas NEMs showed a
bimodal distribution with about 60% of these cells expres-
sing this antigen. Moreover, approximately 30% of NEMs
expressed variable levels of MHC class II, whereas these
molecules were absent from most of the other Nrp1þand
Nrp1cells. Finally, most NEMs were negative for integrin
a2 (CD49b), which instead showed a bimodal distribution in
Gr1Nrp1cells. Supplementary Fig. S1 shows quantifi-
cation of 4 independent experiments.
NEMs were further characterized for the expression of a
number of other cell surface antigens (Supplementary Fig. S2).
These cells were positive for the panleukocytic antigen CD45
and for the FcgRIII receptor CD16, but negative for B220
(expressed by B cells and plasmacytoid dendritic cells) and
for Ly6C (which is also seen by the Gr1 antibody used for
sorting). The cells were also negative for the IL-2 receptor a
chain (CD25), which is usually present on activated T and B
cells and is considered a marker of classical monocyte acti-
vation. NEMs were clearly negative for the Tie2 angiopoietin
receptor (5). Finally, both types of Nrp1þcells produced plexin
A1, A3, B2, B3, C1, and D1; plexins A1, A3, and B2 were
NEMs Induce Tumor Vessel Stabilization
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differentially expressed in Gr1þand Gr1cells (Supplemen-
tary Fig. S3).
Taken together, these results define NEMs as a subset of
Gr1, bone marrow–resident monocytes specifically charac-
terized by the expression of the Nrp1 receptor.
Bone marrow NEMs exert antitumor activity
We moved on to assess the effect of NEMs on established
tumors. Mice (n¼10 per group) were subcutaneously injected
with B16.F10 tumor cells and, at days 11 (when tumors became
palpable) and 13 after inoculation, the tumors were inoculated
with bone marrow–purified NEMs (CD11bþGr1Nrp1þ
cells), CD11bþGr1þNrp1þcells (cells labeled in green and
cyan in Fig. 1, respectively), or PBS. We decided to use the
CD11bþGr1þNrp1þcells (hereafter named Gr1þ)asa
control as these represent a subset of inflammatory monocytes
relatively similar to NEMs, but unresponsive to Sema3A (Figs.
1C and B, respectively).
Cells (2 10
5
cells/tumor/injection; n¼10) or PBS (100 mL)
were delivered directly into the tumor mass. NEM injection
determined a significant reduction in tumor growth (tumor
volume at 15 days: 469 84 in the NEM group vs. 1169 229 in
the Gr1þgroup vs. 806 127 in the PBS-injected mice,
expressed as percentage of pretreatment tumor size; P<
0.01; Fig. 2A). This effect was not consequent to the direct
inhibition of tumor cell proliferation, as the coculture of
purified NEMs or Gr1þcells together with B16.F10 cells did
not affect tumor cell proliferation (Fig. 2B).
A comparably powerful antitumor activity was observed
in a more aggressive, highly metastatic model of tumor
xenograft, obtained by subcutaneous injection of 4T1 cells
in Balb/c mice (n¼10 per group). Bone marrow NEMs,
Gr1þcells, or PBS were administered twice (2 10
5
cells/
tumor/injection) directly into the growing tumor masses.
Again, NEMs inhibited tumor growth (tumor volume at 18
days: 512 94 in the NEM-injected group vs. 1348 776 in
Figure 1. Isolation and characterization of NEMs from the bone marrow. A, sorting of bone marrow NEMs. a, morphological gating of lymphocytes and
monocytes; b, gating of CD11bþcells (on cells gated in a); c, gating for Gr1 expression (on cells gated in b); d, cells analyzed for Nrp1 expression
(on Gr1population in c); e, cells analyzed for Nrp1 expression (on Gr1þpopulation in c); f, schematic representation of the 4 monocyte subpopulations.
Percentages are referred to total bone marrow cells. B, migration assay with the sorted populations; color code as in A, f. FBS was a positive control
(mean SEM; ,P<0.01 over control). C, HCL on gene expression data from the 4 sorted cell populations. Data were internally normalized using the mean
centering method, according to which the mean value of a column (gene expression values of a given gene among samples) is equal to 0. Green, low
expression; red, high expression. D, flow cytometric analysis of the 4 sorted cell populations using the indicated phycoerythrin-conjugated antibodies.
Percentages of positive cells (horizontal bars) relative to total cells are reported.
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the Gr1þ-injected group vs. 1976 1422 in the PBS-injected
mice, expressed as percentage of pretreatment tumor size;
P<0.01; Fig. 2C). Similar to B16.F10 cells, in this case, neither
NEMs nor Gr1þcells affected tumor cell proliferation in
vitro (Fig. 2D).
Ex vivo labeling of NEMs and Gr1þcells with the fluorescent
dye DiD before injection indicated that the cells persisted
inside the tumors at least up to 6 days after inoculation (Fig.
2E).
Staining for the PCNA antigen indicated that there were no
significant differences in the percentage of proliferating B16.
F10 cells in the 3 groups of treated animals (Fig. 2F; quanti-
fication in Fig. 2G); similar results were also obtained for 4T1
tumors (Supplementary Fig. S4).
Figure 2. NEMs exert indirect
antitumor activity. A, growth curve of
B16.F10 melanoma tumors
implanted (day 0) subcutaneously in
C57BL/6 mice and injected at days
11 and 13 with NEMs, Gr1þcells, or
PBS. Data are expressed as
percentages over day 10, mean 
SEM. B, proliferation assay of B16.
F10 cells cocultured in vitro with 2 
10
4
Gr1þcells, NEMs, or PBS after a
2-day coculture (mean SEM). C,
same as in A for 4T1 tumors. D, same
as in B for 4T1 cells. E, fate of sorted
Gr1þand NEM cells injected into
B16.F10 tumors. Before injection,
cells were labeled with DiD (red);
tumors were harvested 6 days after
cell injection. Top (a), whole tumor
sections; bottom (b and c),
magnification of the indicated areas.
Scale bars in the lower panels, 100
mm. F, immunofluorescence of tumor
sections from mice injected with PBS
(a), Gr1þcells (b and b'), or NEMs (c);
green, PCNA; blue, 40, 6-diamidino-
2-phenylindole (DAPI). Asterisk,
vessel lumen; arrow, perivascular,
PCNAþcells. G, percentage of
PCNAþnuclei in the tumor sections
(mean SEM; n¼8 per group).
NEMs Induce Tumor Vessel Stabilization
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Together, these results are consistent with the conclusion
that NEMs markedly inhibit tumor growth when injected
intratumorally, albeit not directly affecting tumor cell
proliferation.
NEMs promote tumor vessel stabilization
The numerous clusters of PCNAþcells detected in corre-
spondence with the tumor vessels (indicated by arrows in Fig.
2F) were possibly indicative of an ongoing angiogenic process.
As these clusters were reduced in the NEM-injected tumors, we
carried out a detailed analysis of the effect of NEMs or Gr1þ
cells on vascular morphology and function. The number of
CD31-positive vessels was not different among the 3 experi-
mental groups (representative images in Fig. 3A and B; quan-
tifications in Fig. 3C and D). However, the vasculature in the
NEM-treated tumors showed markedly increased positivity for
a-SMA and NG2 staining (Figs. 3A and B) and, consequently, a
marked increased in a-SMA/CD31 (P<0.01 vs. both PBS and
Gr1þ) and NG2/CD31 (P<0.05 vs. PBS and P<0.01 vs. Gr1þ)
ratios (Fig. 3E and F, respectively). Morphometric analysis of
the vasculature indicated that, in addition to pericyte coverage,
vessels in the NEM-treated tumors displayed additional mar-
kers of maturation, such as increased mural thickness (Fig. 3G)
and reduced lumen perimeter (Fig. 3H) and area (Fig. 3I).
We also wanted to exclude that the effect of NEMs on vessel
maturation might be a consequence of the reduced tumor
growth caused by their inoculation. Mice (n¼5 per group)
bearing B16.F10 tumors were injected at days 11 and 13 with 2
10
5
NEMs or Gr1þcells, or PBS, and sacrificed when tumors
reached the fixed volume of 1,700 mm
3
. Also in this experiment,
a significant difference in a-SMA/CD31 ratio was evident (P<
0.01 vs. both PBS and Gr1þ; Supplementary Fig. S5).
Collectively, these results indicate that the antitumoral
effect of NEMs is concomitant with a marked increase in
vessel maturation.
NEMs enhance tumor perfusion and relieve tumor-
associated hypoxia
Recent evidence suggests that the establishment of a chronic
state of hypoxia might fuel tumor growth (25, 26). We, there-
fore, wanted to assess whether the vessel stabilization activity
of NEMs might improve tumor perfusion, thus alleviating
Figure 3. NEMs promote vessel maturation. A, immunofluorescence of the vasculature of tumors injected with PBS, Gr1þcells, or NEMs. Whole tumo r
sections were stained for CD31 (green) and a-SMA (red) at 3 different magnifications (a, b, c, d, g, j, m, p, and s). Split CD31 and a-SMA signals are shown in the
small panels e, f, h, i, k, l, n, o, q, r, t, and u under the respective merged pictures. Blue, DAPI. Scale bars in panel d and o, 100 mm. B, same as in A,
using antibodies against CD31 (green) and NG2 (red). C–I, quantitative evaluation, in tumors injected with PBS, Gr1þcells, or NEMs, of the following
parameters: vessel number (C), area covered by CD31þendothelial cells (D), a-SMA/CD31 ratio (E), NG2/CD31 ratio (F), mural thickness (G), vessel lumen area
(H), vessel perimeter (I). Data are mean SEM. ,P<0.05; ,P<0.01 versus PBS.
Carrer et al.
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tumor-associated hypoxia. First, a set of animals injected with
B16.F10 melanoma tumors received either bone marrow NEMs
or Gr1þcells (n¼5 per group) and were then submitted to a
modified Miles assay to verify the permeability of the tumor
vasculature. The results of this assay indicated that the tumors
injected with NEMs displayed vessels that were significantly
less leaky than those injected with Gr1þcells (Fig. 4A). Another
set of tumor-bearing animals (n¼5 per group) was injected
with either bone marrow NEMs or control Gr1þcells. After 3
days, a bolus of doxorubicin (10 mg/kg per animal) was
administered to each mouse. After an additional 4 hours, mice
were sacrificed and the doxorubicin content within the tumor
was assessed. NEM-injection significantly improved intratu-
moral drug delivery (Fig. 4B).
Consistent with the above findings, PD echographic visual-
ization of the tumor vasculature revealed a net increase in the
number of Doppler-positive vessels, reflecting an improvement
in functional tumor perfusion (sequential representative
images of NEM- and Gr1þ-injected tumors are shown
in Fig. 4C, quantification in Fig. 4D). Accordingly, by staining
the tumors by HypoxyProbe (n¼5), we found that NEMs
improved tumor oxygenation, resulting in a reduced hypoxic
tumor area (representative images and quantification in Figs.
4E and F, respectively). Finally, the levels of the hypoxia-
sensitive transcription factor HIF-1awere markedly reduced
in whole lysates from these tumors compared with those
injected with Gr1þcells (shown in Fig. 4G for 4 independent
tumors). Concomitant with reduced HIF-1 activation, there
was a significant decrease in the levels of the mRNAs of 3 major
HIF-1a-regulated genes (VEGF-A,Angiopoietin-2, and Il-6;
ref. 27; Supplementary Fig. S6).
Taken together, these observations would indicate that the
antitumor activity of NEMs is mediated by the induction of
morphological maturation and functional normalization of the
tumor vasculature.
Sema3A-recruited NEMs inhibit tumor growth and
stabilize vessels
Recent work indicates that Sema3A exerts antitumoral
effect by affecting the tumor vasculature (28–32). As our
previous work indicated that Sema3A is a powerful attractor
of NEMs in vivo (17), we wanted to assess whether the Sema3A-
recruited NEMs possessed antitumoral activity. An AAV2
vector expressing Sema3A was injected into the tibialis ante-
rior muscle of both legs in mice (n¼15). After 15 days, muscles
were infiltrated by CD11bþNrp1þGr1cells (Fig. 5A). These
mNEM were purified using anti-CD11b–coupled magnetic
beads (>90% purify of CD11bþNrp1þafter recovery; Fig.
5B). Mice (n¼6 per group) injected subcutaneously with
B16.F10 cells received either mNEMs (2 10
5
cells/tumor per
injection) or PBS; cell delivery was carried out twice, at days 10
and 14 after tumor implantation. mNEM injection significantly
Figure 4. NEMs enhance tumor perfusion and relieve tumor-associated hypoxia. A, quantification of vascular permeability in tumors injected with Gr1þcells
or NEMs (mean SEM; ,P<0.01). B, doxorubicin quantification in tumors injected with Gr1þcells or NEMs (mean SEM; ,P<0.05). C, power
Doppler imaging of 2 tumors injected with Gr1þcells or NEMs; 3 sections (frames 1 to 3) per animal are shown. Large vessels appear in orange–yellow.
D, quantification of blood flow by Doppler analysis (mean SEM; ,P<0.01). E and F, analysis of tumor hypoxia by pimonidazole (green) in sections of
tumors injected with NEMs or Gr1 þcells. E, representative pictures; F, quantification (mean SEM; ,P<0.01). G, Western blotting of whole B16.F10 tumor
lysates (WTL) from 4 different mice treated with Gr1þcells or NEMs using anti-HIF-1aand antitubulin antibodies.
NEMs Induce Tumor Vessel Stabilization
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reduced tumor growth (tumor volume at 20 days: 405 80 in
the mNEM group vs. 810 180 in the controls; P<0.01; Fig. 5C).
Similar to bone marrow NEMs, the antitumoral activity of
mNEMs correlated with their capacity to promote tumor vessel
maturation, as evaluated from the extent of pericyte coverage
after staining for CD31 and a-SMA (Fig. 5D and quantified
in Fig. 5E).
Paracrine activity of NEMs toward vascular smooth
muscle cells
To understand the mechanisms by which NEMs were capa-
ble of normalizing the tumor vasculature, and leading from the
reasonable assumption that this effect was exerted in a para-
crine fashion, we analyzed the levels of expression of a series of
secreted factors and their receptors participating in blood
vessel formation. Bone marrow NEMs produced a few chemo-
kines traditionally associated with M1 macrophages and
known to generally exert antitumoral activity (33), such as
CCL2, CCL4, CCL5, CXCL9, and CXCL10, as well as other
different molecules involved in vessel stabilization (such as
PDGFb, TGF-b, SDF-1, and the antiangiogenic molecule
thrombospondin-1; ref. 34; Fig. 6A). In contrast, NEMs did not
express CCL1, CCL17, and CCL22, chemokines traditionally
associated with M2 macrophages, which have proangiogenic
properties (33). A full list of the analyzed molecules is provided
in Supplementary Table S1.
Figure 5. NEMs recovered from
Sema3A-expressing muscles
inhibit tumor growth and promote
vessel maturation. A, cells
infiltrating muscles 15 days after
injection of AAV2-Sema3A.
Hematoxylin staining (a) and
immunofluorescence for CD11b
(b), Nrp1 (c), Gr1 (d), or double
immunofluorescence for Nrp1 and
Gr1 (e, with split signals in f and g).
Nuclei are counterstained with
DAPI; scale bar, 50 mm. B, flow
cytometry of mNEMs. Cells
purified from Sema3A-expressing
muscles were gated for
morphology (top) and analyzed for
expression of CD11b and Nrp1
(bottom). C, growth curve of B16.
F10 melanoma tumors implanted
(day 0) in C57/BL6 mice and
injected with mNEMs (mean 
SEM). D, vessels in tumors treated
with mNEMs or PBS at 2 different
magnifications. Green, CD31; red,
a-SMA; blue, DAPI. Scale bars, 50
mm. E, a-SMA/CD31 ratio in tumors
treated with mNEMs or PBS (mean
SEM; ,P<0.01).
Carrer et al.
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The presence of vessels covered by a-SMA and NG2-positive
cells was the most striking feature of NEM-injected tumors.
Thus, we assessed whether the factors produced by NEMs had
a direct effect on vascular smooth muscle cell (vSMC) prolif-
eration or migration. vSMCs were seeded in a 24-well plate and
cocultured with bone marrow-purified NEMs or Gr1þcells.
The percentage of proliferating cells was assessed 20 hours
after NEM addition. No significant effect was detected on
vSMC proliferation by either cell subset (Fig. 6B). Next, we
tested the migratory capacity of vSMCs upon stimulation by
NEMs or Gr1þcells in a Transwell chamber assay. Notably,
NEMs attracted vSMCs at a significantly greater extent than
Gr1þcells and at levels similar to the stimulation exerted by
FBS (Fig. 6C; P<0.01).
Collectively, these findings support the conclusion that
NEMs exert their function in a paracrine manner by secreting
factors that stimulate vessel maturation and, in particular,
coverage of endothelial cells through the attraction of mural
cells.
Discussion
The existence of a population of myeloid cells that was
specifically chemoattracted by Nrp1 ligands was originally
unveiled by the observations that VEGF-A
165
and Sema3A,
which both bind Nrp1, were able to induce the migration of
bone marrow–derived, CD11bþcells in vitro and in vivo (16–
18). We show here that the subset of CD11bþNrp1þGr1
monocytes (NEMs), purified from the normal bone marrow,
specifically respond to Sema3A chemoattraction and possess
unique properties in terms of cytokine expression and func-
tional activity.
The flow cytometric profile of bone marrow–derived NEMs
displayed several characteristics common with mouse resident
monocytes, including low expression of Gr1/Ly6C and CD14,
high levels of CD16, intermediate levels of F4/80, as well as
expression of low levels of CXCR4 and CD31 (23, 35, 36). Other
studies have indicated that Nrp1 is expressed by subsets of
myeloid cells both in tumors, where they exert proangiogenic
activity (37), and during development (38). In this respect,
however, Nrp1 expression is known to significantly increase
during monocyte maturation (39), whereas this receptor is
expressed at very low levels in the bone marrow (40). Thus,
although Nrp1 expression distinguishes NEMs among bone
marrow Gr1monocytes, expression of this marker might also
be acquired by other cells in different conditions. In any case,
gene expression profiling and surface marker characterization
clearly indicate that NEMs stand apart from other monocyte
populations that exert proangiogenic activity, such as TAMs,
which express a different panel of cytokines from NEMs (41), or
TEMs, which are positive for Tie2 expression (5), whereas
NEMs are clearly negative. In addition, while Sema3A acts as
a chemoattractant for NEMs, it is known to exert an apoptotic
effect for the M2 monocyte/macrophages also expressing
Nrp1 (39).
Figure 6. Effects of NEMs toward
vascular smooth muscle cells. A,
expression of genes involved in
macrophage polarization and vessel
maturation in NEMs (mean SEM
after normalization for a
housekeeping gene). B, EdU-
positive vSMCs upon coculture with
210
4
Gr1þcells or NEMs, or in the
presence of 20% FBS (positive
control; mean SEM). C, vSMCs
migrated in response to NEMs or
Gr1þcells (mean SEM;
,P<0.01).
NEMs Induce Tumor Vessel Stabilization
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Among the bone marrow Nrp1þmonocytes, only the Gr1
fraction (NEMs) was able to migrate in response to Sema3A;
this is consistent with the observation that all the cells infil-
trating the muscle overexpressing Sema3A were Gr1. After
Sema3A stimulation, Nrp1 signals through heterodimerization
with 1 of the 4 type A plexins, which serve as signal-transducing
elements (42, 43). Gene expression analysis indicated that the
levels of plexin A1 and A3 were higher in NEMs compared with
Gr1þcells, possibly suggesting that these 2 receptors are
involved in the response to Sema3A. Further experiments are
clearly required to verify this possibility.
Once inoculated in vivo, NEMs persisted in the injected
tumors and markedly inhibited tumor growth in the 2 inves-
tigated tumor models. These cells were found to express
several factors involved in vessel maturation (including
PDGFb, TGF-b, and thrombospondin; ref. 44). The vasculature
of the tumors injected with NEMs showed increased pericyte
coverage and mural thickness, more regular shape, reduced
leakiness, and, most importantly, improved function. This is
also consistent with the observation that NEMs selectively
produced factors able to chemoattract vSMCs. In vivo, the
ultimate outcome of these effects was the improvement of
tumor mass perfusion and reduction of tumor hypoxia, which
is a major determinant of tumor progression (13). These
morphological and functional parameters fit the concept of
vessel normalization, as originally put forward as a goal to
improve blood supply and enhance delivery of chemothera-
peutic drugs by low-dose antiangiogenic agents (14, 45).
We and others have shown that Sema3A functions as potent
antiangiogenic factors and inhibits tumor development (28–
32). In particular, the ectopic delivery of an AAV vector
expressing Sema3A into RipTag2 mice significantly reduced
vascular density and branching, inhibited tumor growth and
metastasis, and substantially extended survival (28, 29). Con-
sistent with the possibility that the recruitment of NEMs might,
at least in part, be responsible for the effects of Sema3A, we
found that the cells recruited by Sema3A in vivo actually
exerted significant antitumoral activity. Thus, although no
evidence exists at this moment to show that NEMs might play
a physiological role during normal tumor development, they
might mediate the effects of Sema3A treatment on tumor
growth. In line with this concept, bone marrow NEM inoc-
ulation can be conceived as an experimental treatment able
to normalize the tumor vasculature. The vascular phenotype
obtained by the inoculation of NEMs, at least in the inves-
tigated tumor models, resembles that observed through a
variety of genetic approaches, including the depletion of
endothelial HIF-1a(27), the overexpression of the histidine
rich glycoprotein to skew the TAM response toward an
M1 phenotype (25), or the haploinsuffiency of endothelial
PHD2 (46). In addition, vascular normalization was observed
upon administration of anti-PlGF antibodies (26). All these
conditions are known to improve tumor oxygenation and
lessen tumor malignancy and invasiveness.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: A. Carrer, S. Moimas, S. Zacchigna, G. Serini,
E. Giraudo, F. Bussolino, M. Giacca
Development of methodology: A. Carrer, S. Moimas, S. Zacchigna, M.
Sinigaglia
Acquisition of data: S. Moimas, L. Pattarini, L. Zentilin, G. Ruozi, M. Mano,
M. Sinigaglia
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): A. Carrer, S. Moimas, S. Zacchigna
Writing, review, and/or revision of the manuscript: A. Carrer, S. Moimas,
G. Serini, E. Giraudo, F. Bussolino, M. Giacca
Administrative, technical, or material support (i.e., reporting or orga-
nizing data, constructing databases): F. Maione
Study supervision: S. Zacchigna, M. Giacca
Acknowledgments
The authors thank Mauro Sturnega for assistance in animal experimentation,
Paolo Maiuri for immunofluorescence analysis, and Suzanne Kerbavcic for
editorial work.
Grant Support
Financial support for this work was provided by grants from the "Fondazione
CR Trieste," Trieste, Italy, the FIRB RBAP11Z4Z9 project, and the European
Research Council (ERC) Advanced Grant 250124 FunSel to M. Giacca, and the
Associazione Italiana per la Ricerca sul Cancro (AIRC) to S. Zacchigna, G. Serini,
E. Giraudo, and F. Bussolino.
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 February 28, 2012; revised September 5, 2012; accepted September 5,
2012; published OnlineFirst December 7, 2012.
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