ARTICLES

CSF-1R inhibition alters macrophage polarization and

blocks glioma progression

npg

2013 Nature America, Inc. All rights reserved.

Stephanie M Pyonteck1,6, Leila Akkari1,6, Alberto J Schuhmacher1,6, Robert L Bowman1, Lisa Sevenich1,
Daniela F Quail1, Oakley C Olson1, Marsha L Quick1, Jason T Huse2,3, Virginia Teijeiro1, Manu Setty4,
Christina S Leslie4, Yoko Oei5, Alicia Pedraza2, Jianan Zhang2,3, Cameron W Brennan2,3, James C Sutton5,
Eric C Holland1,3, Dylan Daniel5 & Johanna A Joyce1,3
Glioblastoma multiforme (GBM) comprises several molecular subtypes, including proneural GBM. Most therapeutic approaches
targeting glioma cells have failed. An alternative strategy is to target cells in the glioma microenvironment, such as tumorassociated macrophages and microglia (TAMs). Macrophages depend on colony stimulating factor-1 (CSF-1) for differentiation
and survival. We used an inhibitor of the CSF-1 receptor (CSF-1R) to target TAMs in a mouse proneural GBM model, which
significantly increased survival and regressed established tumors. CSF-1R blockade additionally slowed intracranial growth
of patient-derived glioma xenografts. Surprisingly, TAMs were not depleted in treated mice. Instead, glioma-secreted factors,
including granulocyte-macrophage CSF (GM-CSF) and interferon-g (IFN-g), facilitated TAM survival in the context of CSF-1R
inhibition. Expression of alternatively activated M2 markers decreased in surviving TAMs, which is consistent with impaired
tumor-promoting functions. These gene signatures were associated with enhanced survival in patients with proneural GBM. Our
results identify TAMs as a promising therapeutic target for proneural gliomas and establish the translational potential of CSF-1R
inhibition for GBM.
GBM, the most aggressive form of glioma, has an invariably terminal
prognosis, as patients respond minimally to currently used therapies, including surgery, radiation and chemotherapy1. One challenge
in treating GBM is substantial tumor-cell and genetic heterogeneity, leading to aberrant activation of multiple signaling pathways2,3.
Underscoring this heterogeneity was the identification of several GBM
molecular subtypes: proneural, neural, mesenchymal and classical 4.
In contrast, noncancerous stromal cells in the tumor microenvironment are genetically stable therapeutic targets5,6. TAMs in particular
are associated with high tumor grade and poor prognosis in many
cancers, including gliomas79. A paracrine CSF-1epidermal growth
factor (EGF) signaling loop has also been implicated in breast cancer
and glioma invasion10,11.
Several approaches have been used to ablate TAMs or inhibit their
tumor-promoting functions in mouse models of cancer12. One strategy is CSF-1R inhibition, which depletes macrophages and reduces
tumor volume in several xenograft models13,14. Here we used a potent,
selective CSF-1R inhibitor in multiple preclinical GBM models. These
included RCAS-hPDGFB (the human platelet-derived growth factor
B gene)/nestinTv-a; Cdkn2a/ transgenic mice15,16, referred to here
as PDGF-Bdriven glioma (PDG) mice, and intracranial xenografts of
either proneural GBM cell lines or patient-derived tumor spheres. We
found that CSF-1R inhibition blocks glioma growth and progression

through a mechanism in which TAMs are not depleted but are instead
re-educated within the glioma microenvironment.
RESULTS
Macrophages are targets of CSF-1R inhibition in PDG GBMs
In PDG mice, nestin+ glial progenitors express the RCAS virus receptor, Tv-a, which enables specific delivery of the initiating oncogene,
PDGFB. PDG tumorigenesis recapitulates pathological and molecular
features of human proneural GBM4,1719. We first investigated whether
macrophages accumulate in PDG GBMs as in human gliomas8,9.
Indeed, the numbers of CD11b+ myeloid cells and macrophages were
markedly increased in PDG GBMs compared to normal brain, and
the mRNA expression of Cd68, Csf1 and Csf1r was elevated in the
PDGs (Supplementary Fig. 1a,b). Glioma cells and TAMs express
Csf1, whereas Csf1r is only expressed in macrophages (Fig. 1a,b and
Supplementary Fig. 1ce).
BLZ945 is a brain-penetrant inhibitor of CSF-1R (Supplementary
Fig. 2a and Supplementary Methods). The biochemical half-maximum
inhibitory concentration (IC50) for CSF-1R is 1 nM, which is >3,200fold higher than its affinity for other kinases (Supplementary Table 1).
Treatment of wild-type (WT) bone marrowderived macrophages
(BMDMs) with BLZ945 inhibited CSF-1dependent proliferation (half-maximum effective concentration (EC50) = 67 nM) and

1Cancer

Biology and Genetics, Memorial Sloan-Kettering Cancer Center (MSKCC), New York, New York, USA. 2Human Oncology and Pathogenesis Program, MSKCC,
New York, New York, USA. 3Brain Tumor Center, MSKCC, New York, New York, USA. 4Computational Biology, MSKCC, New York, New York, USA. 5Novartis
Institutes for Biomedical Research, Emeryville, California, USA. 6These authors contributed equally to this work. Correspondence should be addressed to
J.A.J. (joycej@mskcc.org).
Received 31 December 2012; accepted 14 August 2013; published online 22 September 2013; doi:10.1038/nm.3337

1264

VOLUME 19 | NUMBER 10 | OCTOBER 2013

NATURE MEDICINE

npg

Live cells

DAPI

CSF-1R

GBM

DAPI CSF-1R CD68

4
3
Normal brain

2
1
0

Tv-a

Csf1

BMDM

2.0
1.5
1.0

*** ***
***

0.5
0
0

2
3
Time (d)

Csf1r

PDGC-21

4.0

PDGC-31

3.0
2.0
1.0
0
0

2
3
Time (d)

No CSF-1
Vehicle
67 nM BLZ945
670 nM BLZ945
6,700 nM BLZ945

4.0
3.0
2.0
1.0
0
0

2
3
Time (d)

Dose once per day

Initiate tumors with

RCAS-hPDGFB
(56 weeks of age, t = 0)

Population doubling

Cd11b

Population doubling

Relative mRNA level

Macrophages

1
Vehicle or 200 mg kg BLZ945
treatment started
(t = 2.5 weeks after tumor initiation)

Treatment
started

Defined
endpoint

Kill when symptomatic

or at defined endpoint
(t = 26 weeks)

No glioma
Grade II
Grade III
Grade IV/GBM

100

***

100

50

25

BLZ945
Vehicle

P = 1 107

Percentage of mice

75

80
60
40
20

0
0

4 6 8 10 12 14 16 18 20 22 24 26 28
Time after RCAS-hPDGFB-HA injection (weeks)

decreased CSF-1R phosphorylation, similarly to blockade with

CSF-1Rspecific antibody (Fig. 1c and Supplementary Fig. 2bd).
BLZ945 also reduced the viability of CRL-2467 microglia, Cdkn2a/
BMDMs (from the PDG genetic background) and BMDMs from nonobese diabetic severe combined immunodeficient (NOD-SCID) mice
(Supplementary Fig. 3ac). Notably, BLZ945 treatment in culture
did not affect the proliferation of any PDG-derived tumor-cell lines
(all of which were Csf1r negative) or U-87 human malignant glioma
(MG) cells, and PDG cell tumor-sphere formation was unaffected
(Fig. 1d and Supplementary Fig. 3dg). Thus, BLZ945 has no direct
effects on glioma cells and perturbs macrophage survival through
CSF-1R inhibition.
CSF-1R inhibition blocks glioma progression and improves
survival
We next assessed BLZ945 in preclinical trials using PDG mice. At
2.5 weeks after glioma initiation, 30% of the animals had small
tumors (grade II or III) and 70% had no tumors by histology or
magnetic resonance imaging (MRI) (Supplementary Fig. 4a,b).
We treated mice with BLZ945 or vehicle and evaluated them for
symptom-free survival (Fig. 1e). The median survival time in the

NATURE MEDICINE

Glioma cells

Population doubling

Figure 1 CSF-1R inhibition specifically targets

macrophages, improves survival and decreases
glioma malignancy in the transgenic PDG model.
(a) Expression of Csf1 and Csf1r in different cell
populations from GFP-expressing PDGs: a mixed
population of live cells (DAPI), purified glioma
cells (GFP+) and macrophages (CD11b+Gr-1).
Cd11b and Tv-a were used as cell typespecific
control genes for macrophages and glioma cells,
respectively. Expression is shown relative to that
in the live cell fraction and was normalized to
Ubc for each sample (n = 3 mice per group).
(b) Representative immunofluorescence images
of normal brain or GBM PDGs co-stained with
CSF-1R, CD68 (for macrophages and microglia)
and DAPI. Scale bars, 50 Mm. (c) Graph showing
that the CSF-1R inhibitor BLZ945 blocked BMDM
survival, with an effect comparable to that of
CSF-1 deprivation, assessed by MTT assays.
n = 13 independent replicates. (d) Graph showing
MTT assays for BLZ945 treatment of independent
PDGF-driven glioma cell (PDGC) primary lines
derived from PDG mice (Supplementary Figs. 3d
and 7c). Concentrations up to 6,700 nM BLZ945
(100 times the dose required to effectively kill
BMDMs in vitro) had no effect on glioma cell
survival or proliferation. n = 3 independent
replicates. (e) Experimental design for the longterm survival trial: PDG mice were injected with
RCAShPDGFBhemagglutinin (HA) between 5
and 6 weeks of age to induce glioma formation and
were assigned randomly to vehicle (20% Captisol,
n = 22) or BLZ945 (200 mg per kg body weight
(mg kg1), n = 14) treatment groups at 2.5 weeks
after injection. Mice were dosed once daily until
they developed symptoms or reached the trial
endpoint. (f) Symptom-free survival curves for
the mice described in e. (g) Histological grading
of the vehicle (n = 14) and BLZ945 (n = 13)
treatment groups. The data in a, c and d are shown
as the mean o s.e.m. Statistical significance was
calculated by unpaired two-tailed Students
t test (c,d), log-rank (Mantel-Cox) test (f) or Fishers
exact test (g). *P < 0.05, ***P < 0.001.

Percentage symptom-free survival

2013 Nature America, Inc. All rights reserved.

ARTICLES

VOLUME 19 | NUMBER 10 | OCTOBER 2013

0
Vehicle

BLZ945

vehicle-treated cohort was 5.7 weeks. In contrast, BLZ945 treatment

significantly improved long-term survival, with 64.3% of the treated
mice surviving to the trial endpoint at 26 weeks (Fig. 1f). We chose
this endpoint because Cdkn2a/ mice develop spontaneous tumors,
including lymphomas and sarcomas, beginning at ~30 weeks 20.
BLZ945 was well tolerated over the long-term treatment with no
visible side effects (Supplementary Fig. 4e), which is consistent with
results from previous histopathological studies 21. Histological grading revealed high-grade, invasive gliomas in all vehicle-treated mice.
In contrast, BLZ945-treated animals had tumors that were significantly less malignant, and there were no detectable lesions in 55.6%
of the mice that were asymptomatic at the trial endpoint (Fig. 1g and
Supplementary Fig. 4c,d,f).
We then monitored the effects of BLZ945 on established high-grade
PDGs, which are histologically similar to human GBMs at time of
diagnosis17. We performed a 7-d trial incorporating MRI to assess initial tumor volume and subsequent growth (Fig. 2a). We randomized
PDG mice with detectable tumors (4.540 mm3) into BLZ945 or vehicle treatment cohorts. In the vehicle treatment group, tumor growth
increased substantially. In contrast, BLZ945 halted glioma growth,
and the majority of tumors decreased in size in the mice treated with

1265

ARTICLES
a

1 d

6d

Dose once per day

Tumor volume (mm )

**
2 1 0

900
800
700
600
500
400
300
200
100
0
Mouse number 1

1 2 3 4 5
Treatment day (d)

Vehicle

9 10 11

BLZ945

**

100

***

75
50
25
0

Percentage difference in
tumor volume (1 d to 6 d)

Tumor volume (mm3)

50
25

Trial
endpoint

Treatment
started
125

***

75

Percentage difference in
tumor volume (1 d to 6 d)

2013 Nature America, Inc. All rights reserved.

npg

c
Vehicle
BLZ945
BLZ945 large

100

Follow-up MRI
(t = 23 d, 6 d)

Trial
endpoint

Treatment
started

125

Treatment
started
(t = 0 d)

2 1 0

1 2 3 4 5
Treatment day (d)

BLZ945

125
100
75
50
25
0
25
50

75
Mouse number 1

BLZ945 large

MRI for tumor volume

(45 weeks after
tumor initiation; t = 1 d)

Percentage difference in
tumor volume (1 d to 6 d)

Initiate tumors with

RCAS-hPDGFB
(56 weeks of age)

Vehicle

Euthanize animals at
trial midpoint or endpoint
(t = 3 d, 7 d)

9 10 11

25

BLZ945 large

0
25
50

75
Mouse number 1 2 3 4 5 6 7 8 9 1011 12 13 14 15 16 17 18

Figure 2 CSF-1R inhibition blocks tumor growth and effectively regresses established gliomas. (a) Experimental design for the short-term 7 d trial: PDG
mice bearing detectable tumors by MRI were randomly assigned to the vehicle or BLZ945 treatment group, with follow-up MRI as shown. (b,c) Mean
tumor volume over the time course of the experiment for mice whose starting tumor volume was 4.540 mm 3 (vehicle or BLZ945, n = 11 per group; b)
or >40 mm3 (BLZ945 large, n = 18; c). (d) Representative images of T2-weighted MRI scans from the start and endpoint of the trial. The dashed lines
indicate the regions of interest used to calculate tumor volume. (e) Waterfall plots showing the change in tumor volume at the experimental endpoint
relative to the starting tumor volume for each individual mouse. Horizontal dashed lines indicate a 30% decrease in tumor volume. In the vehicletreated group, there was a progressive, substantial increase in tumor growth ranging from 195% to 879%. In contrast, in this short treatment period,
6 of 18 mice in the BLZ945 large group had >30% reduction in tumor volume, qualifying as a partial response according to the response evaluation
criteria in solid tumors (RECIST). The data in b and c are shown as the mean o s.e.m. Statistical significance was calculated by unpaired two-tailed
Students t test. **P < 0.01, ***P < 0.001.

BLZ945 (Fig. 2b,d,e and Supplementary Fig. 5a,b). We then studied

a third cohort of mice with large gliomas (>40 mm3) that we treated
with BLZ945, and the vast majority of these mice showed regression
(Fig. 2ce and Supplementary Fig. 5c). Comparison with a sizematched vehicle-treated cohort was not possible, as most of those
mice would not have survived to the trial endpoint. We confirmed
inhibition of CSF-1R phosphorylation in all BLZ945-treated tumors,
and hPDGF-B production by glioma cells was unaffected by treatment
with BLZ945 (Supplementary Fig. 6).
Because we used a PDGF-driven model for these initial trials, we
wanted to exclude possible off-target effects of BLZ945 on glioma PDGF
receptor (PDGFR) signaling. Glioma cells express PDGFR-A, and pericytes express PDGFR-B (Supplementary Fig. 7a,b). PDGFR inhibition
reduces U-87 MG cell viability22,23. PDG tumors were modestly responsive to PDGFR inhibition in vivo24 and in culture (Supplementary
Fig. 7c). The affinity of BLZ945 for PDGFR-A is approximately
10,000-fold lower than for CSF-1R (Supplementary Table 1).
Unlike PDGFR inhibitors, treatment with BLZ945 or an antibody

1266

to CSF-1R did not affect the viability of the U-87 MG or PDG cell
lines (Fig. 1d and Supplementary Figs. 3de and 7c). Collectively
these data establish that the therapeutic effects of BLZ945 in vivo are
through CSF-1R inhibition and not off-target inhibition of PDGFR.
Multiple hallmarks of cancer are altered by CSF-1R blockade
To determine the mechanisms underlying the marked response to
CSF-1R inhibition in established gliomas, we investigated multiple
tumorigenic processes, including tumor grade, proliferation, angiogenesis and resistance to apoptosis. (Supplementary Table 2).
We performed analyses on tissues from the 7-d BLZ945 trial,
including tissues taken at the midpoint (3 d) and endpoint (7 d)
to capture the dynamic effects of CSF-1R inhibition. Whereas all
vehicle-treated mice had high-grade gliomas, BLZ945-treated
animals exhibited a pronounced histological response that was characterized by tumor-cell depopulation (Fig. 3a and Supplementary
Fig. 8a). Correspondingly, glioma cell numbers within the regions
of the original tumors decreased markedly (Fig. 3bd).

VOLUME 19 | NUMBER 10 | OCTOBER 2013

NATURE MEDICINE

ARTICLES
b

Histological response
(BLZ945)

Vehicle

BLZ945 large

BLZ945
7d

3d

3d

7d

DAPI Brdu Olig2

Grade IV/GBM
(vehicle)

DAPI CC3

2 105
1 105
0

***
**

**
*

3d 7d 3d 7d

npg

2013 Nature America, Inc. All rights reserved.

Vehicle BLZ945 BLZ945 large

120
100
80
60
40
20
0

***
***
**
***

***

3d 7d 3d 7d
Vehicle BLZ945 BLZ945 large

25
20
15
10
5
0

***
***
**
***
NS

3d 7d 3d 7d

Vehicle BLZ945 BLZ945 large

Percentage of
CC3+ cells

3 10

Percentage of
+
+
BrdU Olig2 cells

4 105

Percentage of
Olig2+ cells

Total number
of DAPI+ cells

10
9
8
7
6
5
4
3
2
1
0

**
*
*** *
*

3d 7d 3d 7d
Vehicle BLZ945 BLZ945 large

Figure 3 Short-term BLZ945 treatment results in reduced tumor grade and proliferation and increased apoptosis. (a) Representative H&E images from
the 7-d trial in the PDG model showing a GBM (vehicle treatment) and the histological response to BLZ945. (b) Representative images from the 3-d
and 7-d time points stained for Olig2 (glioma cells), BrdU, CC3 and DAPI. White arrows indicate rare BrdU +Olig2+ cells in the BLZ945-treated groups.
(cf) Quantification of the total number of DAPI+ cells per tumor (c), the percentage of Olig2+ cells relative to the total number of DAPI+ cells (d), the
percentage of proliferating BrdU+Olig2+ cells relative to the total number of DAPI+ cells (e) and the percentage of apoptotic CC3+ cells relative to the
total number of DAPI+ cells (f) (n = 6 for vehicle, n = 5 for 3 d BLZ945, n = 6 for 7 d BLZ945, n = 5 for 3 d BLZ945 large, n = 5 for 7 d BLZ945
large). These analyses revealed a progressive reduction in cell number, and by 7 d, the average glioma cell density was reduced to a20% of the total
cells in both BLZ945 treatment groups. Analysis of glioma cell proliferation revealed a 6798% reduction after BLZ945 treatment. The circles in cf
represent individual mice, and horizontal lines represent the means. Scale bars (a,b), 50 Mm. Statistical significance was calculated by unpaired twotailed Students t test. NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.001.

Proliferation analyses revealed a 6798% reduction in glioma-cell

proliferation after BLZ945 treatment (Fig. 3b,e). In addition, apoptosis increased by 9- to 17-fold after 3 d (Fig. 3b,f), and tumor vascularity decreased. This reduction was probably not due to direct effects of
BLZ945 on endothelial cells, as their viability was unaffected in culture (Supplementary Fig. 8bf). Thus, CSF-1R inhibition effectively
reduces the growth and malignancy of gliomas through the combined
effects of blocking cell proliferation and increasing apoptosis.
CSF-1R inhibition slows intracranial growth of proneural GBM
xenografts
We next asked whether CSF-1R inhibition is effective in experimental
models of human proneural GBM, including primary tumor spheres
(TS573 and TS1137; Supplementary Table 3) and established cell lines
(U251 and LN229) (Supplementary Methods). The tumor spheres and
cell lines were either negative for CSF1R expression or showed negligible
CSF1R mRNA levels compared to the human macrophage line THP-1,
and none of these tumor spheres or GBM cell lines was sensitive to
BLZ945 in monoculture (Fig. 4a,b and Supplementary Fig. 9a,b).
We intracranially injected the glioma tumor sphere or cell lines into
NOD-SCID mice and treated established tumors with either vehicle or
BLZ945. All four human lines responded to BLZ945 in vivo, showing
a clear histological response and significantly reduced tumor growth
and invasion (Fig. 4cf and Supplementary Fig. 9ce). Collectively
these results demonstrate the preclinical efficacy of CSF-1R inhibition across multiple proneural GBM models, including genetically
engineered mice and human-derived xenografts.
Glioma-supplied survival factors protect TAMs from BLZ945induced depletion
Macrophage survival depends on CSF-1R signaling25, and we thus
expected CSF-1R inhibition to deplete macrophage numbers. Indeed,

NATURE MEDICINE

VOLUME 19 | NUMBER 10 | OCTOBER 2013

BLZ945 treatment reduced the number of CD11b+Ly6G cells in the

blood of WT mice. Microglia numbers in normal brains were substantially decreased by BLZ945 treatment; surprisingly, however,
CSF-1R inhibition did not affect TAM numbers in PDGs compared
to vehicle treatment (Fig. 5a,b and Supplementary Figs. 10 and 11).
Similarly, TAMs were not depleted in human tumorsphere and cellline xenografts (Supplementary Fig. 12a,b).
BLZ945-treated TAMs retained CSF-1R expression in vivo, and
there were no significant differences in expression compared to
vehicle-treated PDGs (Supplementary Fig. 13a). We examined
whether the relative proportions of resident microglia and recruited
BMDMs were altered in the PDG model. We performed BM transplantation experiments using GFP+ BM and initiated gliomas after
confirming BM reconstitution (Supplementary Fig. 13b,c). The
proportion of GFP+CD68+ macrophages in gliomas, determined by
immunofluorescence analysis, was unchanged after BLZ945 treatment (Supplementary Fig. 13d), indicating no evident alteration
in peripheral recruitment. We evaluated additional strategies to
deplete macrophages in other tumor types or the brain and found
that although the numbers of macrophages in peripheral tissues or
normal brain decreased, there was no effect on TAM numbers in
PDGs (Supplementary Fig. 14). We analyzed other immune cell
types in gliomas after BLZ945 treatment and found no significant
differences in cell numbers (Supplementary Fig. 13e). In addition,
given varying reports of CSF-1R expression by neurons26,27, we examined neuron numbers in gliomas and normal brain and found no
differences after treatment with BLZ945 (Supplementary Fig. 13f),
which is consistent with our detection of CSF-1R in macrophages only
(Supplementary Fig. 1ce).
To explore the mechanisms by which glioma TAMs are specifically protected from BLZ945-induced death, we investigated
whether glioma-supplied factors support macrophage survival in the

1267

npg

1.0
0.5
0.01

2
1
0

2
3
Time (d)

U251

4
3
2
1
0
0

Vehicle

2
3
Time (d)

BLZ945

250
50

***

25
0
14

***
0

5
10
Time (d)

DAPI

GFP

Vehicle
BLZ945

6,700 nM BLZ945

TS573

U
25

Treatment
started
TS573

500

67 nM BLZ945

15

Vehicle

BLZ945

Treatment
started
250

Vehicle
BLZ945

U251

200

DAPI

GFP

Tumor cell
inoculation

150

50

0
7

***

5
10
Time (d)

***
15

H&E

100

Tumor cell
inoculation

presence of CSF-1R inhibition. Indeed, glioma cellconditioned

medium (GCM) from PDGs or human cell lines protected BMDMs
from death induced by BLZ945 (Fig. 5c and Supplementary
Figs. 12c and 15a,b). This was analogous to the TAM maintenance that we observed for all of our in vivo models (Fig. 5a and
Supplementary Figs. 11, 12a and 13d) and also indicated that the
survival factors were secreted. We screened GCM from a panel
of PDG cell lines and identified multiple protective lines and one
nonprotective line (Supplementary Fig. 16a). By comparing the
GCM from the protective lines to that from the nonprotective line
using antibody arrays, we identified differentially secreted proteins (Supplementary Fig. 16b,c), which we tested in parallel with
known macrophage survival factors28. From this screen, we identified three cytokines that promoted the survival, proliferation or
both of BMDMs in the presence of BLZ945: GM-CSF (also called
CSF-2), IFN-G and chemokine (C-X-C motif ) ligand 10 (CXCL10)
(Fig. 5d,e and Supplementary Figs. 17 and 18ad). Notably, when
we added these factors in combination to GCM from the nonprotective line, it conferred protection from BLZ945-induced killing to the
BMDMs (Supplementary Fig. 18e) in association with elevated Akt
activation in macrophages, similarly to treatment with GCM from
protective lines (Fig. 5f,g). Consistent with their protective effect,
expression of these cytokines and their receptors was substantially
elevated in PDGs compared to normal brain and were also produced by TAMs themselves (Supplementary Fig. 19). These results
establish that glioma-supplied factors promote TAM survival in the
presence of a CSF-1R inhibitor.

1268

Population doubling

CSF1R expression

Vehicle
670 nM BLZ945

Population doubling

TH
P1
TS
57
3

Relative mRNA level

Relative tumor growth

Figure 4 BLZ945 inhibits orthotopic tumor growth

of patient-derived proneural tumor spheres and
cell lines in vivo. (a) CSF1R mRNA expression
in human proneural tumor sphere cells (TS573)
and in the human proneural cell line U251
compared to the human macrophage line THP-1
(positive control). Expression is normalized to
B2M (also called B2M) for each sample. n = 3
independent replicates. (b) MTT assays of BLZ945
treatment tested in TS573 and U251 human
glioma cells demonstrating no effect of BLZ945 at
concentrations up to 6,700 nM. n = 3 independent
replicates. (c,d) Relative tumor growth determined
by bioluminescence imaging (BLI) output in
NOD-SCID mice injected intracranially with
5 104 TS573 cells (c) or 2.5 105 U251 cells (d).
Treatment with BLZ945 was initiated when tumors
were in a positive growth phase determined by BLI,
which corresponded to day 14 for TS573 cells and
day 7 for U251 cells. Mice were randomly assigned
to vehicle (n = 12 for TS573, n = 16 for U251)
or BLZ945 (n = 11 for TS573, n = 17 for U251)
treatment groups. Tumor growth was evaluated
every 5 d for 15 d, at which point vehicle-treated
mice became symptomatic and both cohorts
were euthanized for further analyses.
(e,f) Representative images of the vehicle-treated
and BLZ945-treated TS573 (e) and U251 (f)
xenograft tumors described in c and d stained for
GFP (tumor cells) and DAPI. Representative H&E
images of both treatment groups for the U251
xenografts are shown in the bottom row in f. Scale
bars (e,f), 50 Mm. The data in ad are shown as
the mean o s.e.m. Statistical significance was
calculated by nonparametric two-tailed MannWhitney test. *P < 0.05, **P < 0.01, ***P < 0.001.

Relative tumor growth

2013 Nature America, Inc. All rights reserved.

ARTICLES

CSF-1R inhibition reduces M2 macrophage polarization in

treated gliomas
We next asked how CSF-1R inhibition elicited such a potent antitumor
response in vivo despite no evident TAM depletion. We hypothesized
that TAMs from CSF-1R inhibitortreated animals were functionally
altered, and we used microarray expression profiling of TAMs isolated
from BLZ945- and vehicle-treated gliomas to identify 257 differentially
expressed genes. 52 genes were significantly upregulated and 205 were
downregulated; together this is referred to as the total gene signature
(Fig. 6a, Supplementary Fig. 20a,b and Supplementary Table 4). CSF1R inhibition in BLZ945-treated TAMs was corroborated by downregulation of targets of early growth receptor 2 (Egr2), a transcription factor
that is located downstream of CSF-1R29 (Supplementary Fig. 20c).
To determine the minimal set of genes that discriminated the treatment groups, we employed a lasso logistic regression model30 (Online
Methods). This method identified a five-gene signature for BLZ945
treatment: Adm (adrenomedullin), Arg1 (arginase 1), F13a1 (encoding
a clotting factor), Mrc1 (mannose receptor C type 1, also called Cd206),
Serpinb2 (encoding a protease inhibitor and also called Pai2) (Fig. 6b);
this is referred to as the minimal gene signature. Of these genes, four
were downregulated after BLZ945 treatment. Quantitative PCR analyses on whole gliomas and sorted cell populations confirmed these findings (Supplementary Fig. 20d,e). Serpinb2, the only upregulated gene
in the minimal signature, correlates positively with increased survival
in breast cancer31. Interestingly, each of the downregulated genes has
been associated previously with alternatively activated/M2 macrophage
polarization3237 (Supplementary Table 4).

VOLUME 19 | NUMBER 10 | OCTOBER 2013

NATURE MEDICINE

ARTICLES
a

Adjacent brain
DAPI CD68

Glioma
DAPI CD68

Mean number of CD11b+

cells per 63 FOV per mouse

Figure 5 CSF-1R inhibition depletes normal

BLZ945
Vehicle
microglia but not TAMs in treated PDGs as a
3d
7d
18
result of the production of glioma-supplied
16
survival factors. (a) Representative images of
14
PDGs (top) and adjacent normal brain from the
12
contralateral hemisphere of tumor-bearing mice
10
8
(bottom) from the 7 d BLZ945 trial stained for
6
CD68 and DAPI. White arrows indicate CD68+
4
macrophages in adjacent normal brain from
2
0
both the vehicle- and BLZ945-treated groups.
3d
7d
3d
7d
Scale bars, 50 Mm. (b) Quantification of the
BLZ945 large
Vehicle
BLZ945
+
mean number of CD11b macrophages per 63
field of view (FOV) within each mouse tumor
GCM + vehicle
Vehicle
Vehicle
of the different treatment groups. (c) Graph
DMEM + BLZ945
CXCL10
GM-CSF
4
GCM + 67 nM BLZ945
showing MTT assays of BMDMs demonstrating
NS
4
4
NS
CXCL1
IFN-
GCM + 670 nM BLZ945
that GCM induced BMDM proliferation and
NS
VEGF-A
3
***
3
3
***
**
protected BMDMs from BLZ945-induced
NS
***
2
2
apoptosis. n = 19 independent replicates.
NS
2
NS
For comparison, BMDMs were cultured in
1
1
1
nonconditioned medium supplemented with
0
0
0
CSF-1. (d,e) Results compiled from MTT
2
3
0
1
4
BLZ945
BLZ945
Vehicle
Vehicle
assays demonstrating that GM-CSF and IFN-G
Time (d)
individually protected BMDMs against BLZ945*
induced death (n = 9 independent replicates; d),
**
1.5
*
***
whereas CXCL10 promoted proliferation (n = 6
***
*
independent replicates; e). These effects were
1.0
*
*
**
not reproduced by other candidate factors,
0.5
for example, CXCL1 and vascular endothelial
pAkt
0
(Ser473)
growth factor A (VEGF-A), which are shown
Total Akt
here and in Supplementary Figure 17.
(f) Western blots showing activation of Akt
BLZ945
BLZ945
(Ser473 phosphorylation site) in BMDMs.
BMDMs were stimulated with freshly prepared
GCM from protective (PDGC-23, PDGC-17 and
BLZ945
PDGC-02) or nonprotective (PDGC-55) cell lines
with or without BLZ945. In addition, GCM from
nonprotective PDGC-55 cells was supplemented with survival factors (GM-CSF, IFN-G and CXCL10) with or without BLZ945. (g) Quantification of pAkt
normalized to total Akt demonstrating significant changes in pAkt levels between BMDMs stimulated with the protective compared to the nonprotective
GCM with or without BLZ945. n = 4 independent replicates. RU, relative units. BLZ945 was used at 670 nM in all cell culture assays unless otherwise
specified. The circles in b represent individual mice (n = 7 for vehicle, n = 5 for 3 d BLZ945, n = 5 for 7 d BLZ945, n = 5 for 3 d BLZ945 large, n = 5
for 7 d BLZ945 large), and horizontal lines represent the means. The data in c, d, e and g are shown as the mean o s.e.m. Statistical significance was
calculated by unpaired two-tailed Students t test. NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.001. There were no significant differences for
any of the comparisons performed in b.
Population doubling

SF
PD -1
G
PD C2
PDGC 3 G
G -17 CM
PD C-0 G
G 2 C
C C GCM
SF -5
PD -1 5 G M
C
G
M
PD C2
PDGC 3 G
C
1
PDGC 7 G M
G -02 C
C G M
P 5
+ DG 5 GCM
co C C
m -5 M
P bi 5
+ DG ned GC
co C f M
m -5 ac
bi 5 to
ne G rs
d CM
fa
ct
or
s
pAkt normalized
to total Akt (RU)

PD
C
PDGC SF
-2 PDGC 3 C 1
-1
PDGC 7 CM
+
co P GC-02 M
m D -5 CM
bi G 5
ne C- C
d 55 M
fa C
c M
PD tors
G
PD C CS
-2 F
PDGC 3 C-1
G -17 M
P
D C C
+
co P GC-02 M
m D -5 C
bi G 5 M
ne C- C
d 55 M
fa C
ct M
or
s

2013 Nature America, Inc. All rights reserved.

***
***

npg

***

Population doubling

Population doubling

In many tumors, TAMs are M2 polarized, which is associated with

protumorigenic functions38. Further, macrophages in human gliomas
exhibit an M2-like phenotype, which is correlated with higher tumor
grade9. Given the striking M2 gene enrichment in the minimal signature, we examined the total gene list to determine whether other
M2-associated markers were similarly downregulated by BLZ945,
which revealed five additional genes (Supplementary Fig. 20f and
Supplementary Table 4). Apart from interkeukin-1B (IL-1B), classically activated/M1 genes were not correspondingly upregulated
(Supplementary Fig. 20g). Interestingly, CSF-1 favors M2 macrophage polarization in culture39,40, which is consistent with our finding
that BLZ945 reduces M2 gene expression in vivo.
To explore how glioma cells and CSF-1R inhibition regulate M2
macrophage polarization, we used cell-based assays in which we
exposed BMDMs to GCM to model microenvironmental interactions. Expression of all nine differentially expressed M2 genes
(Supplementary Fig. 20f) significantly increased, and these
increases were reversed by BLZ945 (Fig. 6c). Analysis of a subset of
these genes in two microglia cell lines, BV-2 and CRL-2467, showed
similar results (Supplementary Fig. 21a,b). We next examined
Mrc1 expression in cocultures and freshly isolated mouse glioma
microenvironment cultures containing TAMs. We selected Mrc1

NATURE MEDICINE

VOLUME 19 | NUMBER 10 | OCTOBER 2013

because it is a well-established M2 flow cytometry marker, which

facilitated the determination of its macrophage-specific expression
in mixed cultures. Mrc1 expression also decreased in response to
BLZ945 (Fig. 6d and Supplementary Fig. 21c,d), paralleling its
downregulation in vivo.
Interestingly, human GCM suppressed macrophage phagocytosis
in vitro in conjunction with M2 polarization41. To determine whether
BLZ945 altered macrophage phagocytosis in vivo, we co-stained tissues for the macrophage cell-surface marker CD11b, the glioma cell
marker Olig2 (ref. 42) and cleaved caspase 3 (CC3). Phagocytic capacity (number of CD11b+ macrophages that had engulfed Olig2+CC3+
glioma cells), increased 7.1-fold in the group of PDG mice with large
gliomas treated with BLZ945 compared to mice treated with vehicle
(Supplementary Fig. 22 and Supplementary Table 2). To control for
enhanced glioma cell death, we analyzed phagocytic capacity after
irradiation and found that it increased fourfold. Therefore, macrophages in BLZ945-treated gliomas undergo an increase in phagocytic
function that is apparently not dependent solely on enhanced tumor
apoptosis. Together these data suggest that after CSF-1R inhibition,
glioma TAMs lose M2 polarization and show enhanced phagocytosis, providing a molecular corollary for their impaired tumorpromoting functions.

1269

ARTICLES
a

BL
Z
BL 94
Z 5
BL 94 6
Z 5
BL 94 1
Z 5
BL 94 7
Z 5
BL 94 5
Z 5
BL 94 8
Z 5
BL 94 4
Z 5
Ve 945 2
h
Ve icl 3
h e
Ve icl 7
h e
Ve icl 5
h e
Ve icl 6
h e
Ve icl 2
h e
Ve icl 1
h e
Ve icl 8
hi e 3
cl
e
4

log10 P

Color key
Figure 6 CSF-1R inhibition impairs heterotypic
2 1 0 1 2
signaling between macrophages and glioma cells
Z-score
and is predictive of improved survival in patients
205
52
10
F13a1
with proneural GBM. (a) Volcano plot showing
8
Adm
Mrc1
257 differentially expressed genes in TAMs
Mrc1
6
from BLZ945-treated mice compared to TAMs
Adm
4
Serpinb2
Arginase 1
from vehicle-treated controls (7-d treatment,
F13a1
2
n = 8 mice per group), which is termed the total
0
Arg1
gene signature. 205 downregulated and 52
12 10 8 6 4 2
2
4
6
8
Serpinb2
Fold change (vehicle/BLZ945)
upregulated genes were differentially expressed
in TAMs in the BLZ945-treated group. (b) Heat
**
map of the minimal gene signature determined
DMEM
**
using a lasso logistic regression model trained
GCM
30
25
**
GCM + BLZ945
on the expression data in a. This analysis
20
1,500
**
15
***
identified five genes, which when considered
***
8
***
together accurately differentiated between the
1,000
***
*
**
**
*
*
*
*
6
BLZ945 and vehicle treatment groups.
**
*** *** ** ***
***
*
500
(c,d) Graph of the expression of M2-associated
4
genes in cultured BMDMs demonstrating an
0
2
increase in expression with exposure to GCM
that was blocked in the presence of BLZ945
0
as determined by quantitative PCR for a panel
Adm Arg1 Cd163 Cdh1 F13a1Hmox1 Il1r2 Mrc1 Stab1
BLZ945
of M2-associated genes identified in the total
GCM
30
gene signature (Supplementary Fig. 20f) (c) or
***
***
GCM + + + + +
flow cytometry for Mrc1 (d). n = 7 independent
Naive BMDM CM + + + + +
Stim BMDM CM + + + + +
20
replicates. (e) Glioma cell-cycle analyses in
Stim BMDM + BLZ945 CM + + + + +
which glioma cells were cocultured with either
pAkt
10
naive-BMDMs or stimulated (stim) BMDMs
pErk
GAPDH
0
(treated with GCM before coculture, as shown
in Supplementary Fig. 15a). Cocultures were
1h
2h
12 h
24 h
48 h
treated with BLZ945 or vehicle and subjected
+ naive
+ stim
to flow cytometric analyses. n = 8 independent
BMDMs BMDMs
replicates. GC, glioma cells. (f) Western blots
BLZ945-like class
showing pAkt (Ser473) and pErk (Thr202/
100
Proneural ***
TCGA
Classical
Tyr204) levels in glioma cells at the time points
Vehicle-like class
data set Mesenchymal
Neural
indicated. Glioma cells were exposed to CM
50
**
Proneural
P = 5.4 106
produced by naive or stimulated BMDMs with or
Combined
Classical
data sets Mesenchymal
without BLZ945 (schematic in Supplementary
Neural
0
Fig. 23a). BLZ945 was used at 670 nM in all
0.1
1
10
0
20
40
60
80
100 120
Hazard ratio
cell culture assays unless otherwise specified.
Time (months)
(g) Patients with proneural GBM in TCGA were
classified into BLZ945-like and vehicle-like classes using the minimal gene signature from b (Supplementary Methods). Patients classified as BLZ945
like showed an increased median survival of 10 months. (h) Hazard ratios and 95% confidence intervals for the minimal gene signature determined
for each subtype of the TCGA and combined data sets. Hazard ratios with a confidence interval that did not cross 1.0 were considered significant.
The data in ce are shown as the mean o s.e.m. Statistical significance was calculated by unpaired two-tailed Students t test (ce), log-rank test (g) or
Walds test (h). *P < 0.05, **P < 0.01, ***P < 0.001.

Ve

hi

cl
e
IL
Ve 4
hi
cl
e
67
nM
67
0
nM

Relative mRNA level

Mean intensity of Mrc1

npg

Percentage of glioma
cells in S phase

al GC
on
Ve e
hi
cl
BL e
Z9
4
Ve 5
hi
cl
BL e
Z9
45

Percentage survival

2013 Nature America, Inc. All rights reserved.

CSF-1R inhibition abrogates macrophage gliomacell

heterotypic signaling
As BLZ945 blocked PDG proliferation in vivo, we examined whether
glioma-cell proliferation was modulated by exposure to macrophages
in cell culture and whether it was reduced by CSF-1R inhibition. We
stimulated WT BMDMs with GCM to mimic their education in
the glioma microenvironment (referred to hereafter as stimulated
BMDMs) and subsequently added them to glioma cells in coculture
(Supplementary Fig. 15a). Cell-cycle analysis showed that coculture
of stimulated BMDMs with PDG cells markedly increased their proliferation. Notably, this induction was abrogated by the addition of
BLZ945 or a CSF-1Rspecific antibody (Fig. 6e and Supplementary
Fig. 15c), suggesting the M2-dependent nature of this effect.
We investigated whether macrophage-secreted factors influence
the activation of known signaling effectors of proliferation and
survival within glioma cells, including Akt and extracellular
signalingrelated kinase (Erk). Levels of phosphorylated Akt (pAkt)
in glioma cells increased in response to CM from the stimulated

1270

BMDMs compared to CM from naive BMDMs. This induction was

abrogated when we collected the CM from stimulated BMDMs in the
presence of BLZ945 (Fig. 6f and Supplementary Fig. 23a,b). pErk
levels in glioma cells did not change during this time course (Fig. 6f).
Addition of the Akt inhibitor MK2206 markedly impaired the macrophage CMinduced proliferation of glioma cells (Supplementary
Fig. 23c), which implicates this pathway as an important driver
of signaling in the GBM microenvironment. We also investigated
whether macrophages promoted glioma-cell proliferation in vivo
by orthotopically co-injecting BMDMs with PDG cells or injecting PDG cells alone. Notably, co-injection of macrophages with
glioma cells enhanced tumor growth by 62% compared to injection
of glioma cells alone, and this induction was blocked by treatment
with BLZ945 (Supplementary Fig. 23d; P < 0.05). Collectively these
data demonstrate that macrophages and glioma cells have reciprocal effects on the survival, proliferation and/or polarization of each
other to promote tumorigenesis, and this heterotypic signaling can
be blocked by CSF-1R inhibition.

VOLUME 19 | NUMBER 10 | OCTOBER 2013

NATURE MEDICINE

npg

2013 Nature America, Inc. All rights reserved.

ARTICLES
Gene signatures of CSF-1R inhibition predict survival advantage
in proneural GBM
We next examined whether BLZ945 gene signatures generated from
mouse TAMs might have translational value through differential survival associations in individuals with GBM. We used the total gene
signature to classify subjects in The Cancer Genome Atlas (TCGA)43
and a second combined series4446 into BLZ945-like and vehicle-like
classes. Notably, there was a proneural-specific increase in median
survival for subjects in the BLZ945-like class that ranged from 7.5 to
31.5 months (Supplementary Fig. 24a,b). We also used the minimal
gene signature and observed that this markedly smaller gene set recapitulated the BLZ945-like survival advantage in subjects with proneural GBM (Fig. 6g,h, Supplementary Fig. 24ad and Supplementary
Tables 5 and 6). Notably, this survival increase was not evident in
other GBM subtypes and was independent of glioma CpG island
methylator phenotype (G-CIMP) status47 (Supplementary Fig. 24e
and Supplementary Table 7).
To determine whether the minimal gene signature predicted survival independently of macrophage number, we used expression
of macrophage-specific genes (AIF1, CD68 and ITGAM) as surrogates for macrophage number in a Cox proportional hazards model
(Supplementary Tables 8 and 9). None of the macrophage-specific
genes correlated with worse prognosis, indicating that the minimal
gene signature of BLZ945 treatment is a subtype-specific, prognostic
predictor of the survival of patients with GBM that is independent of total macrophage number. This also suggests that the TAM
phenotype within a tumor may predict overall survival better than
TAM number per se. The subtype specificity for this survival difference is consistent with the minimal and total gene signatures having
been generated from the PDG model, which most closely represents
proneural GBM19.
DISCUSSION
We show here that a new CSF-1R inhibitor blocks malignant progression, regresses established gliomas and markedly enhances survival
in a transgenic mouse model of proneural GBM. Moreover, multiple
proneural GBM human xenografts responded similarly to CSF-1R
inhibition, underscoring the therapeutic relevance of these findings.
Surprisingly, we found that TAMs in all models were specifically
protected from CSF-1R inhibitorinduced death. This contrasted
with the observed depletion of microglia in the normal brain and
the depletion of macrophages in other tissues, which is consistent
with previous reports13,14. These results suggest that glioma-supplied
factors facilitate TAM survival in the presence of BLZ945, whereas
neighboring microglia outside of the tumor mass are not exposed
to these protective signals and are thus depleted. Modeling these
microenvironment-mediated effects in culture allowed us to screen
and identify GM-CSF and IFN-G as glioma-supplied factors that
facilitate macrophage survival in the context of CSF-1R inhibition.
Notably, expression of these factors (or their receptors) was enriched
in gliomas compared to normal brain, which is consistent with their
prosurvival functions. This led us to ask whether TAM functions
were impaired after CSF-1R inhibition, given that their numbers were
not altered. Indeed, expression analyses of surviving TAMs in vivo
revealed a decrease in alternatively activated M2 macrophage markers, which is consistent with blunted tumor-promoting functions.
We also modeled this phenomenon in culture and demonstrated
that induction of M2 polarization by glioma-secreted factors and its
reversal by CSF-1R inhibition are both direct effects on macrophages.
Together these data indicate that the presence of survival factors in

NATURE MEDICINE

VOLUME 19 | NUMBER 10 | OCTOBER 2013

the glioma microenvironment protects TAMs from CSF-1R inhibitor

mediated killing, enabling their re-education and an associated
marked antitumor response.
There are several potential clinical implications of these findings.
First, macrophage accumulation correlates with malignancy in human
gliomas8,9, which supports therapeutic targeting of TAMs. Second, we
found that alteration of TAM tumor-promoting functions can significantly abrogate malignancy and that depletion is not strictly necessary
for effective macrophage-targeted therapy. Third, in light of our preclinical data in proneural human xenografts and mouse models and
the prognostic advantage associated with BLZ945-like signatures in
subjects with proneural GBM, it is possible that proneural GBMs are
particularly dependent on the tumor-promoting functions of TAMs.
As such, it will be interesting to determine in future studies whether
other GBM subtypes respond similarly to CSF-1R inhibition. Fourth,
myeloid cells, including macrophages, blunt the chemotherapeutic
response in breast cancer models48,49 and promote revascularization
after irradiation in GBM xenografts50. Thus, it may be instructive to
consider CSF-1R inhibitors in combination with glioma celldirected
therapies, opening the possibility for synergistic effects.
In sum, we uncover a new therapeutic strategy for targeting cells
in the glioma microenvironment. Rather than depleting stromal
cells, as has been the goal with many microenvironment-targeted
therapies thus far, re-educating these cells has the potential to not
only abolish their protumorigenic functions but also actively enlist
them as suppressors of tumorigenesis.
METHODS
Methods and any associated references are available in the online
version of the paper.
Note: Any Supplementary Information and Source Data files are available in the
online version of the paper.
ACKNOWLEDGMENTS
We thank K. Simpson and X. Chen for excellent technical support, members of
the Joyce and Holland labs, particularly T. Ozawa, K. Pitter and M. Squatrito, for
technical advice and reagents and D. Chakravarti and the MSKCC Brain Tumor
Center for assistance with patient and tumor sphere line information. We thank
R. Benezra, K. Hunter and H.-W. Wang for reading the manuscript. We thank
E. Pamer and T. Hohl (MSKCC) for providing CCR2-DTR mice and R. Lang
(Cincinnati Childrens Hospital Medical Center) for providing CD11b-DTR mice.
We are grateful to the MSKCC Core Facilities of Genomics, Flow Cytometry
and Small Animal Imaging, Geoffrey Beene Translational Oncology and the
Novartis Institutes for BioMedical Research Emeryville Analytical Sciences group
for technical assistance. This research was supported by US National Cancer
Institute program grants of the Integrative Cancer Biology Program (CA148967;
J.A.J. and C.S.L.) and Tumor Microenvironment Network (CA126518; J.A.J. and
E.C.H.), Cycle for Survival (J.A.J.), the Geoffrey Beene Foundation (J.A.J., R.L.B.
and O.C.O.), the MSKCC Brain Tumor Center (J.A.J. and L.A.), the Fundacin
Ramn Areces and Ibercaja (A.J.S.), Deutsche Forschungsgemeinschaft (L.S.),
Canadian Institutes of Health Research (D.F.Q.), US National Institutes of Health
T32 Institutional Research training grant (5T32GM008539, S.M.P.), US National
Cancer Institute F31 fellowships (F31CA167863, R.L.B.; F31CA171384, O.C.O.)
and Cornell and Gerstner Sloan Kettering graduate programs (S.M.P., R.L.B.,
O.C.O. and V.T.).
AUTHOR CONTRIBUTIONS
S.M.P., L.A., A.J.S., L.S., D.F.Q. and O.C.O. performed and analyzed experiments.
R.L.B., M.S. and C.S.L. performed computational analyses. M.L.Q., V.T., Y.O., A.P.
and J.Z. provided technical assistance or derived patient tumor sphere lines. J.T.H.
performed histopathological analyses. C.W.B., J.C.S., E.C.H. and D.D. provided
reagents. J.A.J. conceived, designed and supervised the study and wrote the
manuscript. All authors edited or commented on the manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.

1271

ARTICLES

npg

2013 Nature America, Inc. All rights reserved.

Reprints and permissions information is available online at http://www.nature.com/

reprints/index.html.

1. Stupp, R. et al. Radiotherapy plus concomitant and adjuvant temozolomide for

glioblastoma. N. Engl. J. Med. 352, 987996 (2005).
2. Wen, P.Y. & Kesari, S. Malignant gliomas in adults. N. Engl. J. Med. 359, 492507
(2008).
3. Dunn, G.P. et al. Emerging insights into the molecular and cellular basis of
glioblastoma. Genes Dev. 26, 756784 (2012).
4. Verhaak, R.G. et al. Integrated genomic analysis identifies clinically relevant
subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR,
and NF1. Cancer Cell 17, 98110 (2010).
5. Balkwill, F.R. & Mantovani, A. Cancer-related inflammation: common themes and
therapeutic opportunities. Semin. Cancer Biol. 22, 3340 (2012).
6. Joyce, J.A. & Pollard, J.W. Microenvironmental regulation of metastasis. Nat. Rev.
Cancer 9, 239252 (2009).
7. Bingle, L., Brown, N.J. & Lewis, C.E. The role of tumour-associated macrophages
in tumour progression: implications for new anticancer therapies. J. Pathol. 196,
254265 (2002).
8. Hussain, S.F. et al. The role of human glioma-infiltrating microglia/macrophages in
mediating antitumor immune responses. Neuro-oncol. 8, 261279 (2006).
9. Komohara, Y., Ohnishi, K., Kuratsu, J. & Takeya, M. Possible involvement of the
M2 anti-inflammatory macrophage phenotype in growth of human gliomas. J. Pathol.
216, 1524 (2008).
10. Wyckoff, J. et al. A paracrine loop between tumor cells and macrophages is required
for tumor cell migration in mammary tumors. Cancer Res. 64, 70227029 (2004).
11. Coniglio, S.J. et al. Microglial stimulation of glioblastoma invasion involves EGFR
and CSF-1R signaling. Mol. Med. 18, 519527 (2012).
12. Ruffell, B., Affara, N.I. & Coussens, L.M. Differential macrophage programming in
the tumor microenvironment. Trends Immunol. 33, 119126 (2012).
13. Manthey, C.L. et al. JNJ-28312141, a novel orally active colony-stimulating factor1 receptor/FMS-related receptor tyrosine kinase-3 receptor tyrosine kinase inhibitor
with potential utility in solid tumors, bone metastases, and acute myeloid leukemia.
Mol. Cancer Ther. 8, 31513161 (2009).
14. Patel, S. & Player, M.R. Colony-stimulating factor-1 receptor inhibitors for the
treatment of cancer and inflammatory disease. Curr. Top. Med. Chem. 9, 599610
(2009).
15. Fomchenko, E.I. et al. Recruited cells can become transformed and overtake PDGFinduced murine gliomas in vivo during tumor progression. PLoS ONE 6, e20605
(2011).
16. Hambardzumyan, D., Amankulor, N.M., Helmy, K.Y., Becher, O.J. & Holland, E.C.
Modeling adult gliomas using RCAS/t-va technology. Transl. Oncol. 2, 8995
(2009).
17. Huse, J.T. & Holland, E.C. Genetically engineered mouse models of brain cancer
and the promise of preclinical testing. Brain Pathol. 19, 132143 (2009).
18. Kennedy, B.C. et al. Dynamics of central and peripheral immunomodulation in a
murine glioma model. BMC Immunol. 10, 11 (2009).
19. Hambardzumyan, D., Parada, L.F., Holland, E.C. & Charest, A. Genetic modeling of
gliomas in mice: new tools to tackle old problems. Glia 59, 11551168 (2011).
20. Serrano, M. et al. Role of the INK4a locus in tumor suppression and cell mortality.
Cell 85, 2737 (1996).
21. Wang, T. et al. Investigation of correlation among safety biomarkers in serum,
histopathological examination, and toxicogenomics. Int. J. Toxicol. 30, 300312
(2011).
22. Kilic, T. et al. Intracranial inhibition of platelet-derived growth factor
mediated glioblastoma cell growth by an orally active kinase inhibitor of the
2-phenylaminopyrimidine class. Cancer Res. 60, 51435150 (2000).
23. Takeuchi, H., Kanzawa, T., Kondo, Y. & Kondo, S. Inhibition of platelet-derived
growth factor signalling induces autophagy in malignant glioma cells. Br. J. Cancer
90, 10691075 (2004).
24. Shih, A.H. et al. Dose-dependent effects of platelet-derived growth factor-B on glial
tumorigenesis. Cancer Res. 64, 47834789 (2004).
25. Chitu, V. & Stanley, E.R. Colony-stimulating factor-1 in immunity and inflammation.
Curr. Opin. Immunol. 18, 3948 (2006).

1272

26. Luo, J. et al. Colony-stimulating factor 1 receptor (CSF1R) signaling in injured

neurons facilitates protection and survival. J. Exp. Med. 210, 157172 (2013).
27. Erblich, B., Zhu, L., Etgen, A.M., Dobrenis, K. & Pollard, J.W. Absence of colony
stimulation factor-1 receptor results in loss of microglia, disrupted brain development
and olfactory deficits. PLoS ONE 6, e26317 (2011).
28. Lin, H. et al. Discovery of a cytokine and its receptor by functional screening of
the extracellular proteome. Science 320, 807811 (2008).
29. Bradley, E.W., Ruan, M.M. & Oursler, M.J. Novel pro-survival functions of the
Kruppel-like transcription factor Egr2 in promotion of macrophage colony-stimulating
factormediated osteoclast survival downstream of the MEK/ERK pathway. J. Biol.
Chem. 283, 80558064 (2008).
30. Friedman, J., Hastie, T. & Tibshirani, R. Regularization paths for generalized linear
models via coordinate descent. J. Stat. Softw. 33, 122 (2010).
31. Croucher, D.R., Saunders, D.N., Lobov, S. & Ranson, M. Revisiting the biological
roles of PAI2 (SERPINB2) in cancer. Nat. Rev. Cancer 8, 535545 (2008).
32. Biswas, S.K. & Mantovani, A. Macrophage plasticity and interaction with lymphocyte
subsets: cancer as a paradigm. Nat. Immunol. 11, 889896 (2010).
33. Trcsik, D. et al. Factor XIII-A is involved in the regulation of gene expression in
alternatively activated human macrophages. Thromb. Haemost. 104, 709717
(2010).
34. Probst-Cousin, S., Rickert, C.H. & Gullotta, F. Factor XIIIa-immunoreactivity in
tumors of the central nervous system. Clin. Neuropathol. 17, 7984 (1998).
35. Chen, P. et al. Tumor-associated macrophages promote angiogenesis and melanoma
growth via adrenomedullin in a paracrine and autocrine manner. Clin. Cancer Res.
17, 72307239 (2011).
36. Schroder, W.A., Major, L. & Suhrbier, A. The role of SerpinB2 in immunity. Crit.
Rev. Immunol. 31, 1530 (2011).
37. Solinas, G. et al. Tumor-conditioned macrophages secrete migration-stimulating
factor: a new marker for M2-polarization, influencing tumor cell motility. J. Immunol.
185, 642652 (2010).
38. Sica, A., Schioppa, T., Mantovani, A. & Allavena, P. Tumour-associated macrophages
are a distinct M2 polarised population promoting tumour progression: potential
targets of anti-cancer therapy. Eur. J. Cancer 42, 717727 (2006).
39. Fleetwood, A.J., Lawrence, T., Hamilton, J.A. & Cook, A.D. Granulocyte-macrophage
colony-stimulating factor (CSF) and macrophage CSF-dependent macrophage phenotypes
display differences in cytokine profiles and transcription factor activities: implications
for CSF blockade in inflammation. J. Immunol. 178, 52455252 (2007).
40. Sierra-Filardi, E. et al. Activin A skews macrophage polarization by promoting a
proinflammatory phenotype and inhibiting the acquisition of anti-inflammatory
macrophage markers. Blood 117, 50925101 (2011).
41. Wu, A. et al. Glioma cancer stem cells induce immunosuppressive macrophages/
microglia. Neuro-oncol. 12, 11131125 (2010).
42. Squatrito, M. et al. Loss of ATM/Chk2/p53 pathway components accelerates tumor
development and contributes to radiation resistance in gliomas. Cancer Cell 18,
619629 (2010).
43. Cancer Genome Atlas Research Network. Comprehensive genomic characterization
defines human glioblastoma genes and core pathways. Nature 455, 10611068
(2008).
44. Freije, W.A. et al. Gene expression profiling of gliomas strongly predicts survival.
Cancer Res. 64, 65036510 (2004).
45. Phillips, H.S. et al. Molecular subclasses of high-grade glioma predict prognosis,
delineate a pattern of disease progression, and resemble stages in neurogenesis.
Cancer Cell 9, 157173 (2006).
46. Murat, A. et al. Stem cellrelated self-renewal signature and high epidermal
growth factor receptor expression associated with resistance to concomitant
chemoradiotherapy in glioblastoma. J. Clin. Oncol. 26, 30153024 (2008).
47. Noushmehr, H. et al. Identification of a CpG island methylator phenotype that
defines a distinct subgroup of glioma. Cancer Cell 17, 510522 (2010).
48. DeNardo, D.G. et al. Leukocyte complexity predicts breast cancer survival and
functionally regulates response to chemotherapy. Cancer Discovery 1, 5467
(2011).
49. Shree, T. et al. Macrophages and cathepsin proteases blunt chemotherapeutic
response in breast cancer. Genes Dev. 25, 24652479 (2011).
50. Kioi, M. et al. Inhibition of vasculogenesis, but not angiogenesis, prevents the
recurrence of glioblastoma after irradiation in mice. J. Clin. Invest. 120, 694705
(2010).

VOLUME 19 | NUMBER 10 | OCTOBER 2013

NATURE MEDICINE

ONLINE METHODS

Mice. All animal studies were approved by the Institutional Animal Care
and Use Committee of MSKCC under protocol 04-08-022. The nestinTv-a;
Cdkn2a (Ink4a/Arf)/ mouse model51,52 (mixed strain background), transgenic CCR2diphtheria toxin receptor (DTR)-CFP mice (C57BL/6)53 (referred
to here as CCR2-DTR) and transgenic CD11b-DTR mice (FVB/N)5456 have
been described previously. WT C57BL/6 and NOD-SCID mice were purchased
from Charles River Laboratories, B-actinGFP (C57BL/6) mice57 were from
Jackson Laboratories, and athymic nude mice were from the National Cancer
Institute Frederick. All lines were bred within the MSKCC animal facility.

npg

2013 Nature America, Inc. All rights reserved.

Culture of established cell lines. The U-87 MG (HTB-14)58 human glioma and
CRL-2467 (ref. 59) mouse microglia cell lines were purchased from ATCC, and
human umbilical vein endothelial cells (HUVECs) and human brain microvascular
endothelial cells (HBMECs) were from Sciencell. The BV-2 (ref. 60) mouse microglia cell line and the U251 and LN229 human glioma cell lines were kind gifts from
E.C.H. All cells were grown at 37 C with 5% CO2, passaged with trypsin and maintained in DMEM, 10% FBS (Gibco) and 1% penicillin-streptomycin (Gibco) unless
otherwise noted. The CRL-2467 cell line was cultured in DMEM and 10% FBS with
30 ng ml1 recombinant mouse CSF-1 (R&D Systems). HUVECs and HBMECs
were cultured on gelatin-coated cell culture dishes in endothelial cell medium
(Sciencell) and 10% FBS supplemented with endothelial cell growth factors.
Culture and characterization of human primary glioma tumor spheres.
Human gliomaderived tumor sphere lines (TS1137 and TS573) were derived
from consenting patients under Institutional Review Board (IRB)-approved
protocols for the banking of excess tumor tissue during routine surgical resection (MSKCC IRB# 99-125A(2) and 06-107), as previously described61. Tumor
spheres were maintained in the human NeuroCult NS-A Proliferation Kit (Stem
Cell Technologies) and were dissociated with Accutase cell detachment solution (Millipore). Characterization and molecular subtyping were performed as
described in the Supplementary Methods.
Thymidine kinaseGFP-luciferase cell labeling. The indicated cell lines (TS573,
TS1137, U251, LN229 and PDGC-23) were labeled with a triple-imaging vector
(thymidine kinase (TK)-GFP-luciferase (Luc) (TGL))62 to allow for noninvasive
in vivo imaging of tumor growth. A standard protocol for retroviral transduction
was used. Briefly, GP2-293T cells were transfected with the TGL construct and
pCL-Ampho at a 1:1 ratio using Fugene (Promega) and OptiMEM (Gibco). 12 h
later, the medium was replaced with complete antibiotic-free DMEM and was
collected for 3 d for the transduction of target cells.
Intracranial injection for tumor initiation in the mouse PDG and human
glioma models. Adult mice were fully anesthetized with ketamine and xylazine
and the local anesthetic bupivacaine at the surgical site before intracranial injection to the right frontal cortex (approximate coordinates: 1.5 mm lateral, 1 mm
caudal from bregma, depth of 23 mm) using a fixed stereotactic apparatus
(Stoelting) as previously described16,24. Additional details are available in the
Supplementary Methods. TGL-labeled human cell lines were resuspended in
antibiotic-free serum-free medium, and 5 104 (TS1137, TS573 and LN229) or
2.5 105 (U251) cells in a volume of 2 Ml were injected into 6-week-old female
NOD-SCID mice. Athymic nude mice (6-week-old females) were injected with
2 Ml of either 5 104 TGL-labeled PDGC-23 cells alone or PDGC-23 cells mixed
with 30% RFP+ BMDMs (1.66 104) in antibiotic-free serum-free medium.
BLZ945 treatment in vitro and in vivo. The CSF-1R inhibitor BLZ945 (supplied
by Novartis) was synthesized as described in the Supplementary Methods. For
cell culture studies, a 10 mM stock of BLZ945 was formulated in DMSO; DMSO
was used as the vehicle control. For administration to mice, BLZ945 was formulated in 20% Captisol at a concentration of 12.5 mg ml1. For in vivo dosing,
mice received 200 mg per kg body weight BLZ945 or vehicle (20% Captisol)
by oral gavage once daily. For long-term survival studies in PDG mice, dosing
was begun at 17 d after injection of RCAShPDGFBHA. For studies at a fixed
time point, PDG mice underwent MRI scans at 45 weeks after injection of
RCAShPDGFBHA, as previously described15. Additional information can
be found in the Supplementary Methods.

doi:10.1038/nm.3337

Immunodeficient (NOD-SCID or athymic nude) animals were orthotopically transplanted with TGL-labeled PDGC-23 cells (in athymic nude mice),
TS573, TS1137, U251 or LN229 cells (in NOD-SCID mice). Mice were randomly
assigned to BLZ945- or vehicle-treatment cohorts starting at the indicated time
points after intracranial injection. Mice were dosed daily for 15 d, and tumor
growth was monitored every 5 d by bioluminescence imaging using a Xenogen
IVIS-200 Optical In vivo Imaging System.
Radiation treatment. Tumor-bearing PDG mice were sedated with ketamine
and xylazine before a single radiation dose of 10 Gy (115 cGy min1)42 using
the X-RAD 320 from Precision X-Ray. Mice were euthanized 24 h after irradiation, and tissues were collected for histological analysis of macrophage
phagocytic capacity.
Bone marrow transplant (BMT). For BMT experiments, nestinTv-a; Cdkn2a/
mice were bred to WT C57BL/6 mice to generate nestinTv-a; Cdkn2a+/
transplant recipients. NestinTv-a; Cdkn2a/ mice were also bred to C57BL/6
B-actinGFP transgenic mice to generate Cdkn2a+/ bone marrow donors that
ubiquitously expressed GFP. For BMT, recipient mice were lethally irradiated
(10 Gy) at 44.5 weeks of age. The next day, donor-mouse bone marrow
was flushed from femurs and tibiae with X-Vivo 20 Media (Cambrex) and
resuspended in sterile PBS at a concentration of 107 cells per ml. Recipients
received 1 106 donor BM nucleated cells in 100 Ml of PBS by intravenous tail
vein injection. After 4 weeks, successful engraftment was assessed by the percentage of GFP+ cells in the blood of BMT recipients by flow cytometry. If engraftment
was less than 60%, mice were excluded from all subsequent experiments.
DT/DTR and clodronate liposome-mediated macrophage depletion.
A detailed description of the experimental procedures can be found in the
Supplementary Methods.
Mouse euthanasia and tissue harvest. Mice were euthanized at the time points
described in the figure legends or when they became symptomatic from their
tumors, which included signs of poor grooming, lethargy, weight loss, hunching,
macrocephaly or seizures. To isolate tissues for snap freezing in liquid nitrogen
or BMDM isolation, mice were euthanized 1 h after the final treatment dose by
CO2 asphyxiation or fully anesthetized with Avertin (2,2,2-tribromoethanol;
Sigma) and cervically dislocated. For flow cytometry, mice were fully anesthetized with Avertin and transcardially perfused with 20 ml of PBS. The brain
was then isolated, and the tumor was macrodissected from the surrounding
normal tissue. For proliferation analysis, mice were injected intraperitoneally
with 100 mg per g body weight of BrdU (Sigma) 2 h before euthanasia. To isolate
tissues for frozen histology, mice were fully anesthetized with Avertin, transcardially perfused with 10 ml of PBS and 10 ml of 4% paraformaldehyde in PBS
(PFA). The brain was post-fixed in PFA overnight at 4 C, and other tissues
were cryopreserved in 30% sucrose at 4 C. After post-fixation, the brain was
transferred to 30% sucrose at 4 C for at least 2 d. All tissues were then embedded in optimal cutting temperature compound (OCT) (Tissue-Tek), and 10-Mm
cryostat tissue sections were used for all analyses.
Preparation of glioma single-cell suspensions. For the derivation of primary
glioma cell cultures or investigation of brain macrophage populations by flow
cytometry, macrodissected PDGs were digested to a single-cell suspension
by 812 min of incubation at 37 C with 5 ml of papain digestion solution
(0.94 mg ml1 papain (Worthington), 0.48 mM EDTA, 0.18 mg ml1 N-acetyll-cysteine (Sigma) and 0.06 mg ml1 DNase I (Sigma) diluted in Earls Balanced
Salt Solution (EBSS) and allowed to activate at room temperature for at least
30 min). After digestion, the enzyme was inactivated by the addition of 2 ml
of 0.71 mg ml1 ovomucoid (Worthington). The cell suspension was passed
through a 40-Mm mesh filter to remove undigested tissue and washed with
Neural Stem Cell (NSC) Basal Medium (Stem Cell Technologies) for subsequent
culture or FACS buffer (1% IgG-free BSA in PBS (Jackson Immunoresearch))
for flow cytometry and centrifuged at a low speed, 750 r.p.m. (Sorvall Legend
RT), to remove debris and obtain the cell pellet.
As many immune-cell epitopes are papain sensitive, for investigation of
immune-cell infiltrates by flow cytometric analysis, tumors were digested to a

NATURE MEDICINE

npg

2013 Nature America, Inc. All rights reserved.

single-cell suspension by incubation for 10 min at 37 C with 5 ml of 1.5 mg ml1

collagenase III (Worthington) and 0.06 mg ml1 DNase I in 1 Hanks Balanced
Salt Solution (HBSS) with calcium and magnesium. The cell suspension was then
washed with PBS and passed through a 40-Mm mesh filter to remove undigested
tissue. To remove myelin debris, the cell pellet was resuspended in 15 ml of 25%
Percoll (room temperature) prepared from stock isotonic Percoll (90% Percoll
(Sigma) and 10% 10 HBSS) and then spun for 15 min at 1,500 r.p.m. (Sorvall
Legend RT) with the accelerator and brake set to 1. The cell pellet was then
washed with 1 HBSS before being resuspended in FACS buffer.
Derivation of mouse primary glioma cultures, tumor spheres and glioma cell
lines. PDG single-cell suspensions were resuspended in DMEM containing 10%
FBS to derive mouse primary glioma cultures, which were used at early passage
(23) and contained a mixture of different cell types, including tumor cells,
macrophages and astrocytes (Supplementary Fig. 21c). For immunostaining,
primary glioma cultures were grown for 24 h on poly-l-lysinecoated coverslips
(BD Biocoat), fixed with PFA overnight at 4 C, permeabilized with 0.1% TritonX for 5 min and blocked with 0.5% PNB (phosphate-NaCl buffer) for at least 1 h
before antibody staining for CD11b+ macrophages (1:200), nestin+ glioma cells
(1:500) and GFAP+ astrocytes (1:1,000) (Supplementary Table 10).
For tumor sphere cultures, the PDG cell pellet was resuspended in mouse
NSC Basal Medium with NSC proliferation supplement, 1 mg ml1 heparin
(Stem Cell Technologies), 10 ng ml1 recombinant human EGF (Invitrogen) and
20 ng ml1 recombinant human basic fibroblast growth factor (FGF) (Sigma).
Fresh medium was added every 72 h for 2 weeks. Primary tumor spheres were
collected, mechanically disaggregated to a single-cell suspension and propagated
by serial passaging. For the secondary tumor sphereformation assay, 5 103
cells were plated in a six-well plate in neurosphere medium in the presence of
BLZ945 or DMSO as a vehicle. The medium was changed every 48 h, and the
number of tumor spheres present after 2 weeks was counted.
To generate PDGC lines, secondary tumor spheres were dissociated to a single-cell suspension and cultivated in DMEM and 10% FBS as a monolayer63.
Multiple glioma cell lines were derived from independent mice, denoted as
PDGC lines. The PDGC-23 line was infected with a pBabe-H2B-mCherry
construct as described previously64.
Isolation of BMDMs. For BM isolation followed by macrophage derivation,
femurs and tibiae were harvested under sterile conditions and flushed. For all
experiments, unless otherwise specified, BM was isolated from WT C57BL/6
mice. The marrow was passed through a 40-Mm strainer and cultured in 30-ml
Teflon bags (PermaLife PL-30) in DMEM and 10% FBS supplemented with
10 ng ml1 recombinant mouse CSF-1 (R&D Systems). BM cells were cultured
in Teflon bags for 7 d with fresh CSF-1containing medium changes every other
day to induce macrophage differentiation, after which they were referred to as
BMDMs.
Histology, immunohistochemistry, flow cytometry and staining analysis.
A detailed description of the experimental procedures can be found in the
Supplementary Methods and in Supplementary Tables 10 and 11.
Preparation of glioma-cell and BMDM CM and heterotypic glioma cell and
BMDM stimulation experiments. CM from PDGC lines or human glioma
cell lines was generated by incubating cells in serum-free DMEM or NeuroCult
medium (for human tumor spheres) for 24 h (Supplementary Fig. 15a).
Collected CM was passed through 0.22-Mm filters to remove cellular debris
and is referred to here as GCM. Undiluted GCM was used to stimulate differentiated C57BL/6 WT or B-actinGFP+ BMDMs without further addition of CSF-1
or other media. Control macrophages received fresh DMEM, 10% FBS and
10 ng ml1 recombinant mouse CSF-1. When indicated, differentiated BMDMs
were cultivated in GCM containing either DMSO as vehicle, 67 nM BLZ945,
670 nM BLZ945 or 8 Mg ml1 of the CSF-1Rneutralizing antibody or in DMEM,
10 ng ml1 mouse recombinant CSF-1 and 10 ng ml1 IL-4 (R&D Systems) for
24 h or 48 h before experimental analysis.
To generate the BMDM CM, three experimental conditions were established
(Supplementary Fig. 23a): (i) serum-free medium and 10 ng ml1 CSF-1
(naive), (ii) GCM and vehicle and (iii) GCM and 670 nM BLZ945, with (ii) and

NATURE MEDICINE

(iii) referred to as the stimulated conditions. The CM from each of these three
treatment groups was then added to serum-starved PDGC-23 glioma cells in
the presence or absence of 200 nM of the Akt inhibitor MK-2206 (ref. 65) before
western blotting or cell-cycle analysis.
Protein array analysis of mouse cytokines and angiogenic factors. Membranes
immobilized with captured antibodies to cytokines and angiogenic factors were
purchased from R&D Systems (from the Cytokine and Angiogenesis Arrays,
respectively) and processed according to the manufacturers instructions. Briefly,
after membranes were blocked for 1 h at room temperature, they were incubated
with 1 ml of each PDGC-derived GCM overnight at 4 C together with the
respective antibody detection cocktails. After extensive washing (three times,
10 min each) to remove unbound factors, membranes were then incubated individually with horseradish peroxidase (HRP)-conjugated streptavidin provided
by the manufacturer for 30 min at room temperature. Unbound materials were
washed out (three times, 10 min each wash). The signal was detected by the
enhanced chemiluminescence (ECL) system provided by the manufacturer.
Cell-cycle analysis. Control BMDMs or BMDMs derived from B-actinGFP+
mice that were prestimulated with GCM were cocultured in a 1:1 ratio with
1 105 serum-starved mCherry-positive PDGC-23 cells for 48 h in the presence
of 670 nM BLZ945, 8 Mg ml1 of the CSF-1Rneutralizing antibody or DMSO as
a vehicle. After the collection of trypsinized cocultured cells, wells were rinsed
in additional medium, and this volume was collected to ensure recovery of all
adherent BMDMs. Samples were washed once with FACS buffer and incubated
for 10 min at room temperature in permeabilizing buffer (10 mM piperazineN,N2-bis(2-ethanesulfonic acid) (PIPES), 0.1 M NaCl, 2 mM MgCl2 and 0.1%
Triton X-100, pH 6.8) containing 0.1 mg ml1 DAPI (Invitrogen). After acquisition on an LSR II flow cytometer (BD) using an ultraviolet laser (350360 nm),
the cell-cycle status of the glioma cells was analyzed using the Flow Jo Dean-JettFox program for cell-cycle analysis. The same procedure was used to analyze the
cell-cycle entry of PDGC-23 cells after exposure to BMDM CM generated from
the three experimental conditions detailed above.
Proliferation assay, protein and RNA isolation, western blotting and quantitative real-time PCR. A detailed description of the experimental procedures
can be found in the Supplementary Methods.
Microarrays and gene expression profiling. All samples were prepared and
processed by the genomics core facility at MSKCC. RNA was isolated using
TRIzol, and the quality was assessed by running on an Agilent Bioanalyzer.
75 ng of total RNA was reverse transcribed and labeled using the Genechip
3` IVT Express Kit (Affymetrix). The resulting cRNA was hybridized to
Affymetrix MOE 430A 2.0 chips. Raw expression data were analyzed using
GCOS 1.4 (Affymetrix). Data were normalized to a target intensity of 500
to account for differences in global chip intensity. The microarray data are
deposited in NCBI Gene Expression Omnibus (GEO) under the accession
number GSE37475.
Microarray analysis. All bioinformatics analyses were completed in R using
the Bioconductor suite of packages. Expression values were computed using
the robust multiarray average (RMA) method and then quantile normalized
in the affy package66,67. The limma package68 was used to identify differentially
expressed genes between the vehicle- and BLZ945-treated samples. Differential
expression was considered significant at a fold change of o2 with a false
discovery rate of 10%. Gene set enrichment analysis (GSEA) was used as
described previously69. For subsequent analyses and comparison to human
data sets, mouse expression values were mean centered across all samples.
Lasso regression method for gene signature identification. Mouse expression data were normalized and mean centered as described above. Differentially
expressed genes were used for further analysis. We trained a logistic regression
model with lasso constraints to differentiate between vehicle- and BLZ945treated samples using the glmnet package30 by setting the family parameter
to binomial in the glmnet function. The regularization parameter for lasso
regression was chosen by fourfold cross validation.

doi:10.1038/nm.3337

Patient glioma data sets, subtyping of non-TCGA patients, human glioma

classification and stratification of patients by G-CIMP status. A detailed
description of the analyses can be found in the Supplementary Methods.
Survival analyses. Survival analyses were completed using the survival package in R70. Hazard ratios were determined using the coxph function from the
survival package. Patients were stratified on the basis of the probability of the
lasso logistic regression classification, G-CIMP status or both, as indicated.
P values were generated using Walds test.

Data presentation and statistical analyses. Data are presented as the means
with their respective standard errors (s.e.m.) or as statistical scatter plots generated using GraphPad Prism Pro5. Within each experiment, independent biological replicates represent a minimum of three technical replicates. Numerical data
were analyzed by unpaired two-tailed Students t test unless otherwise noted.
For survival curves, P values were obtained using the log-rank (Mantel-Cox)
test or the two-tailed Mann-Whitney test when indicated, and Fishers exact
test was used for histological tumor grading. P < 0.05 was considered statistically significant.
51. Dai, C. et al. PDGF autocrine stimulation dedifferentiates cultured astrocytes and
induces oligodendrogliomas and oligoastrocytomas from neural progenitors and
astrocytes in vivo. Genes Dev. 15, 19131925 (2001).
52. Tchougounova, E. et al. Loss of Arf causes tumor progression of PDGFB-induced
oligodendroglioma. Oncogene 26, 62896296 (2007).
53. Hohl, T.M. et al. Inflammatory monocytes facilitate adaptive CD4 T cell responses
during respiratory fungal infection. Cell Host Microbe 6, 470481 (2009).
54. Cailhier, J.F. et al. Conditional macrophage ablation demonstrates that resident
macrophages initiate acute peritoneal inflammation. J. Immunol. 174, 23362342
(2005).

npg

2013 Nature America, Inc. All rights reserved.

Plots for patient analyses. All Kaplan-Meier survival curves, heat maps and
volcano plots were generated in R v 2.14.1 using the gplots and ggplot2 packages71,72. Hazard ratio forest plots were generated in GraphPad Prism Pro5.

55. Duffield, J.S. et al. Selective depletion of macrophages reveals distinct, opposing
roles during liver injury and repair. J. Clin. Invest. 115, 5665 (2005).
56. Ueno, M. et al. Layer V cortical neurons require microglial support for survival
during postnatal development. Nat. Neurosci. 16, 543551 (2013).
57. Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T. & Nishimune, Y. Green mice
as a source of ubiquitous green cells. FEBS Lett. 407, 313319 (1997).
58. Pontn, J. & Macintyre, E.H. Long term culture of normal and neoplastic human
glia. Acta Pathol. Microbiol. Scand. 74, 465486 (1968).
59. Walker, W.S. Establishment of mononuclear phagocyte cell lines. J. Immunol.
Methods 174, 2531 (1994).
60. Bocchini, V. et al. An immortalized cell line expresses properties of activated
microglial cells. J. Neurosci. Res. 31, 616621 (1992).
61. Szerlip, N.J. et al. Intratumoral heterogeneity of receptor tyrosine kinases EGFR
and PDGFRA amplification in glioblastoma defines subpopulations with distinct
growth factor response. Proc. Natl. Acad. Sci. USA 109, 30413046 (2012).
62. Ponomarev, V. et al. A novel triple-modality reporter gene for whole-body fluorescent,
bioluminescent, and nuclear noninvasive imaging. Eur. J. Nucl. Med. Mol. Imaging
31, 740751 (2004).
63. Jensen, J.B. & Parmar, M. Strengths and limitations of the neurosphere culture
system. Mol. Neurobiol. 34, 153161 (2006).
64. Florey, O., Kim, S.E., Sandoval, C.P., Haynes, C.M. & Overholtzer, M. Autophagy
machinery mediates macroendocytic processing and entotic cell death by targeting
single membranes. Nat. Cell Biol. 13, 13351343 (2011).
65. Hirai, H. et al. MK-2206, an allosteric Akt inhibitor, enhances antitumor efficacy
by standard chemotherapeutic agents or molecular targeted drugs in vitro and
in vivo. Mol. Cancer Ther. 9, 19561967 (2010).
66. Irizarry, R.A. et al. Summaries of Affymetrix GeneChip probe level data. Nucleic
Acids Res. 31, e15 (2003).
67. Gautier, L., Cope, L., Bolstad, B.M. & Irizarry, R.A. affyanalysis of Affymetrix
GeneChip data at the probe level. Bioinformatics 20, 307315 (2004).
68. Smyth, G.K. Linear models and empirical bayes methods for assessing differential
expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3, Article3 (2004).
69. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach
for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102,
1554515550 (2005).
70. Therneau, T. A package for survival analysis in S. (R package version 2.3612,
2012).
71. Warnes, G.R. et al. gplots: various R programming tools for plotting data (R package
version 2.10.1, 2011).
72. Wickham, H. ggplot2: Elegant Graphics for Data Analysis (Springer, New York, 2009).

doi:10.1038/nm.3337

NATURE MEDICINE