Neuro-Oncology 1

XX(XX), 1–13, 2019 | doi:10.1093/neuonc/noz080 | Advance Access date 03 May 2019

Genetic and genomic alterations differentially dictate

low-grade glioma growth through cancer stem cell–

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specific chemokine recruitment of T cells and microglia

Xiaofan Guo, Yuan Pan, and David H. Gutmann

Department of Neurology (X.G., Y.P., D.H.G.), Washington University School of Medicine, St Louis, Missouri

Corresponding Author: David H. Gutmann, MD, PhD, Department of Neurology, Box 8111, 660 South Euclid Avenue, Washington
University, St. Louis MO 63110 (gutmannd@wustl.edu).

Abstract
Background. One of the clinical hallmarks of low-grade gliomas (LGGs) arising in children with the neurofibroma-
tosis type 1 (NF1) cancer predisposition syndrome is significant clinical variability with respect to tumor growth,
associated neurologic deficits, and response to therapy. Numerous factors could contribute to this clinical hetero-
geneity, including the tumor cell of origin, the specific germline NF1 gene mutation, and the coexistence of addi-
tional genomic alterations. Since human specimens are rarely acquired, and have proven difficult to maintain in
vitro or as xenografts in vivo, we have developed a series of Nf1 mutant optic glioma mouse strains representing
each of these contributing factors.
Methods. Optic glioma stem cells (o-GSCs) were generated from this collection of Nf1 genetically engineered
mice, and analyzed for their intrinsic growth properties, as well as the production of chemokines that could differ-
entially attract T cells and microglia.
Results. The observed differences in Nf1 optic glioma growth are not the result of cell autonomous growth
properties of o-GSCs, but rather the unique patterns of o-GSC chemokine expression, which differentially attract T
cells and microglia. This immune profile collectively dictates the levels of chemokine C-C ligand 5 (Ccl5) expression,
the key stromal factor that drives murine Nf1 optic glioma growth.
Conclusions. These findings reveal that genetic and genomic alterations create murine LGG biological heteroge-
neity through the differential recruitment of T cells and microglia by o-GSC–produced chemokines, which ulti-
mately determine the expression of stromal factors that drive tumor growth.

Key Points
1.  Differences in LGG proliferation are not dictated by differential o-GSC growth.
2.  Optic GSC–specific chemokine expression differentially attracts T cells and microglia.
3. T cells and microglia collectively specify tumor Ccl5 levels and LGG growth.

One of the hallmarks of many brain tumors is clinical heter- mutation).2–4 Even in children with the low-grade glioma pre-
ogeneity, such that histologically similar pediatric low-grade disposition syndrome, neurofibromatosis type 1 (NF1), the
gliomas (eg, pilocytic astrocytomas [PAs]) can exhibit strik- clinical behavior of the tumors can be dramatically hetero-
ingly different growth patterns and responses to therapy.1 geneous. In this regard, while only 20% of children with NF1
Some of this heterogeneity could result from the causative develop PAs of the optic pathway (optic pathway gliomas),5
genetic mutation (eg, KIAA1549-BRAF alteration versus there are considerable differences between patients with
FGFR1 mutation), tumor location within the neuroaxis (eg, tumors of the same histological subtype.6 This variation
cerebellum versus brainstem), and/or the presence of sec- includes the age of tumor development, the glioma growth
ondary coexisting genomic changes (eg, PTEN or FGFR1 rate, and the response to therapy.

© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved.
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2 Guo et al. Stromal regulation of low-grade glioma growth

Importance of the Study

The factors that contribute to the biological variability gene mutations and secondary genomic alterations.
observed in pediatric LGGs are currently unknown. Using these mice, we demonstrate that the observed

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Defining the etiologies for this clinical heterogeneity differences in overall tumor proliferation do not reflect
is critical for the future implementation of precision the intrinsic cell autonomous growth properties of the
oncology approaches to risk assessment and treat- cancer (optic glioma) stem cells (o-GSCs), but rather
ment for children with these brain tumors. In an effort the differential recruitment of T cells and microglia by
to elucidate the cellular and molecular mechanisms o-GSCs. These findings support a model in which the
that underlie this variability, we employed a series of biological heterogeneity of pediatric LGG is primarily
clinically relevant and authenticated mouse models dictated by stromal cell establishment of a supportive
of Nf1 optic glioma with different germline Nf1 microenvironment.

Defining the individual contributions of these caus-

ative factors to overall glioma biology has proven chal-
lenging in humans, since genomic variation, cell of
Materials and Methods
origin, and tumor microenvironment effects each con- Mice
tribute. Moreover, in the context of NF1, tumors are rarely
biopsied or removed, and the few gliomas obtained All animals were maintained on a C57BL/6 background
have proven difficult to maintain in culture or grow as and used in accordance with an approved Animal Studies
patient-derived xenografts in rodents.7 To define the Committee protocol at Washington University. As previ-
potential molecular and cellular etiologies for pedi- ously described,8–11 we generated Nf1flox/neo; GFAP-Cre (FMC)
atric low-grade glioma heterogeneity, we have lever- mice (Nf1+/− mice with Nf1 gene inactivation in neuroglial
aged Nf1-mutant mouse strains engineered to develop progenitors), Nf1flox/neo; Ptenflox/wt; GFAP-Cre (FMPC) mice
optic gliomas.8–11 Optic gliomas in the setting of NF1 are (additional Pten heterozygous deletion in FMC mice), and
caused by a germline mutation in the NF1 tumor sup- Nf1flox/neo; Olig2-Cre (FMOC) mice (Nf1+/− mice with Nf1
pressor gene coupled with somatic NF1 loss, leading to gene inactivation in Olig2+ precursor cells). Two new strains
biallelic NF1 inactivation.12 Similar to children with NF1, of mice were generated with one allele containing a non-
optic glioma formation in mice with a germline Nf1 gene sense mutation either in exon 10 (c.1149C>A; p.Cys383X) or
mutation occurs following conditional somatic Nf1 loss in in exon 28 (c.3827G>C; p.Arg1278Pro; generously provided
neuroglial progenitors during embryogenesis.8 by Dr Jonathan Epstein, University of Pennsylvania).13
The availability of this experimental platform allows Nf1flox/1149; GFAP-Cre (FM1149C) and Nf1flox/1278; GFAP-Cre
for the introduction of different germline Nf1 gene (FM1278C) mice were produced by intercrossing Nf1+/
mutations,11 the addition of other genomic changes,9 and Cys383X or Nf1+/Arg1278Pro mice with Nf1flox/flox GFAP-Cre mice,

somatic Nf1 loss in different progenitor cell populations.10 respectively. Nf1flox/neo Ccr2+/RFP; GFAP-Cre (FMC Ccr2+/RFP)
Using this strategy, the penetrance of optic gliomas, the mice were generated by intercrossing Nf1flox/neo Ccr2+/RFP
latency to tumor formation, and the level of optic glioma and Nf1flox/flox; GFAP-Cre mice.14
growth can be varied, thus creating a population of genet-
ically engineered mouse strains that more fully capture
the clinical heterogeneity seen in children with NF1–optic Optic Glioma Stem Cells
glioma.
In the current study, a series of low-grade glioma stem Optic nerves/chiasms were dissected from 3-month-
cell preparations (optic glioma stem cells [o-GSCs]), old FMC, FM1149C, FM1278C, and FMPC mice, as well
generated from both previously reported and novel Nf1- as from  6-month-old FMOC mice, and used to generate
mutant mouse strains with optic glioma, were used to o-GSCs.15 Briefly, single cell suspensions were obtained by
demonstrate that mouse optic glioma biological variability digesting the optic nerve/chiasm in Trypsin Digest Medium,
is not accounted for by the intrinsic growth properties of followed by maintenance in neural stem cell (NSC) me-
the cancer cells. Instead, optic glioma proliferation in situ dium (61% Dulbecco’s modified Eagle’s medium [DMEM]
reflects the differential ability of o-GSCs from mice with low glucose, 35% neurobasal medium, 2 mM L-glutamine,
different germline and genomic alterations to produce 1% penicillin/streptomycin) supplemented with epidermal
specific chemokines that recruit T cells and microglia. The growth factor (EGF) (20  ng/mL), fibroblast growth factor
collective action of T cells and microglia then dictates the (FGF) (20 ng/mL), 1% N2, and 2% B-27. For stem cell marker
levels of the key stromal factor (chemokine C-C ligand 5 analysis, o-GSCs were fixed in 4% paraformaldehyde after
[Ccl5]) that drives murine Nf1 optic glioma growth. Taken 24 hours  in culture, and stained with nestin, sex deter-
together, the deployment of these unique strains provided mining region Y–box 2 (sox2), brain lipid binding protein
an experimental system to define differences in the immu- (BLBP), cluster of differentiation (CD)133, or Ki67 antibodies
nologic landscape of pediatric low-grade glioma relevant (Supplementary Table 1). To measure proliferation, o-GSCs
to their biological variability. were incubated with 5-ethynyl-2'-deoxyuridine (EdU) and
 Guo et al. Stromal regulation of low-grade glioma growth 3

Oncology
Neuro-
EdU+ cells using the Click-iT EdU assay (Fisher Scientific). the 96-well Transwell filter (5 µm; Corning) with 50 µL F12-
To measure apoptosis, 105 o-GSCs were plated into 24-well DMEM without FBS. Added to the lower chamber was
plates coated with poly-D-lysine (50 μg/mL) and fibronectin 0.2 mL of o-GSC CM. Microglia on the lower chamber side
(10  μg/mL) in complete NSC medium. After 3  days, cells of the membrane were fixed with 4% paraformaldehyde
were grown in NSC medium without N2, B27, FGF, and 4 hours later, and stained with 4′,6′-diamidino-2-

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EGF for 48 hours, fixed in 4% paraformaldehyde, and ap- phenylindole for quantitation.
optosis quantitated using an in situ cell death detection kit
(Sigma-Aldrich). Self-renewal was assessed using single
o-GSC neurospheres from each mouse strain grown in Western Blotting
complete NSC. After 7 days, the number of resulting sec-
ondary neurospheres was counted, using at least 10 Western blotting was performed as previously reported15
neurospheres per strain. For conditioned medium (CM) using the antibodies in Supplementary Table 1.
experiments, 106 cells were seeded into 6-well plates with
2  mL of NSC medium, which was collected 3  days later.
Immunohistochemistry
Mouse anti-Ccl2 (3  μg/mL, R&D Systems) and anti-Ccl12
(2.5 μg/mL, R&D Systems) antibodies were used for Ccl2/ Mice were euthanized and perfused with Ringer’s solution
Ccl12 neutralizing assays. At least 3 independently iso- and then 4% paraformaldehyde. Optic nerves were paraffin
lated o-GSC preparations per mouse strain were employed embedded, and immunohistochemistry and cell counting
for all experiments. Ras activity in the o-GSC isolates was performed using primary and secondary antibodies
measured using the RAS activation enzyme-linked immu- (Supplementary Table 1).16
nosorbent assay (ELISA) (Cell Biolabs, STA-440), according
to the manufacturer’s instructions.
Chemokine Arrays
T Cells For 3  days, 106 o-GSCs were seeded in plates containing
2  mL of complete NSC. Chemokines in the CM were
Mouse spleens were homogenized in phosphate buffered detected using the Proteome Profiler Mouse Chemokine
saline containing 0.1% bovine serum albumin and 0.6% Array Kit (R&D Systems).
Na-citrate, incubated with 120 units of DNase I  for 15  min
at room temperature, and filtered through a 30-µm strainer.
Single cell suspensions were obtained after red blood cell RNA Extraction and Real-Time Quantitative PCR
lysis (eBioscience, 00433357) and negative selection using the
pan-T-cell isolation kit II (Miltenyi Biotec, 130-095-130). T cells RNA from mouse optic nerves was isolated using Trizol re-
were maintained in Roswell Park Memorial Institute (RPMI)- agent (Life Technologies). RNA from o-GSCs was isolated
1640 medium supplemented with 10% fetal bovine serum using the RNeasy kit (Qiagen). Real-time quantitative re-
(FBS). To assay migration, 105 cells were seeded into the verse transcription (qRT)–PCR was performed16 using spe-
upper chamber of each 24-wellTranswell filter (5 µm; Corning) cific primers (Supplementary Table 2). Values of ΔΔCT were
with 0.1  mL RPMI-1640 without FBS. Added to the lower calculated using H3 as an internal control.
chamber was 0.5 mL of o-GSC CM. After 4 hours, the medium
in the lower chamber was collected, and lymphocytes at the
lower chamber side of the membrane were counted. Where Optic Nerve Measurements
noted, FM1278C or FMPC CM was neutralized with mouse
Mouse optic nerves were microdissected and
anti-Ccl2 antibody (0.04 µg/mL; R&D Systems), mouse anti-
photographed, and the optic nerve diameters measured at
Ccl12 antibody (0.2 µg/mL; R&D Systems), or both, for 1 h at
the chiasm to calculate optic nerve volumes.17
37°C. Activated T cells were obtained by 48 hours CD3 (1 μg/
mL) and CD28 (5 μg/mL) stimulation in vitro. CM from acti-
vated T cells (following CD3/CD28 activation)16 were collected Statistical Analyses
for microglia Ccl5 induction experiments.
All data were analyzed using GraphPad Prism 6 software.
Data between 2 groups were analyzed by unpaired 2-tailed
Microglia Student’s t-tests. Data for multiple comparisons were
analyzed with one-way ANOVA. Each experiment was re-
Mouse brains were homogenized in the gentleMACS
peated at least 3 times. Statistical significance was set at
Dissociator (Miltenyi) for microglia isolation,16 and
P < 0.05.
cells grown in Minimal Essential Medium Earle’s me-
dium supplemented with 1  mM L-glutamine, 1  mM so-
dium pyruvate, 0.6% D-(+)-glucose, 100  μg/mL penicillin/
streptomycin, 4% FBS, and 6% horse serum. After 2 weeks, Results
microglia/macrophages were separated from astrocytes
by shaking (200  g, 5  h, 37°C). Grown in T  cell CM were In order to develop a platform to explore optic glioma
5  ×  105 microglia/macrophages for 24 hours,16 and Ccl5 biological heterogeneity, we generated a collection of Nf1
measured by ELISA (R&D Systems). To assay migration, optic glioma mouse strains that included the original Nf1
6000 microglia were seeded into the upper chamber of optic glioma strain (FMC), in which a neomycin sequence
4 Guo et al. Stromal regulation of low-grade glioma growth

was inserted into exon 31 of the Nf1 gene,8 as well as mice previously shown to regulate optic glioma formation
that differ with respect to the germline Nf1 gene mutation and growth. In this regard, microglia (Iba1+ cells)19–21 and
(c.1149C>A mutation [this report], FM1149C; c.3827G>C T cells (CD3+ cells)16 are required for tumor development
mutation, FM1278C13), the cell of origin (oligodendrocyte and continued growth. As such, we quantified microglia
transcription factor 2 [Olig2+] progenitors, FMOC mice10), (%Iba1+ cells) and T cell (number of CD3+ cells) content in

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and the presence of an additional genomic alteration (Pten each of the murine Nf1 optic gliomas. While FM1149C and
mutation in neuroglial progenitors, FMPC mice9) (Fig. 1A). FM1278C mice did not harbor more microglia than non-
At 3  months of age, FMC, FM1278C, and FMPC mice de- neoplastic (FF) optic nerves, FMPC optic nerves had 2-fold
veloped optic gliomas with increased proliferation (%Ki67+ more microglia than either FMOC or FMC mice (Fig. 3A).
cells; Fig. 1B). Strikingly, FMPC mice exhibit the most While previous studies from our laboratory have shown
proliferative tumors, as previously reported.9 In contrast, that these optic glioma–associated monocytes were
FMOC mice do not develop tumors until 6 months of age.10 CD11b+/CD45low/CX3CR1+ microglia by fluorescence acti-
Lastly, only 20–25% of FM1149C mice (3/12 mice) develop vated cell sorting, we used the microglia-specific marker,
optic gliomas (Supplementary Fig. 1). Tmem119,22 by immunohistochemistry to confirm their
Using this series of Nf1 genetically engineered mice, we identity. Consistent with their designation as microglia, we
first tested the hypothesis that the differences in tumor observed similar patterns and percentages of Tmem119+
proliferation reflected the intrinsic growth properties of cells in the Nf1 optic gliomas (Fig. 3B). Moreover, there
the neoplastic cells in these optic gliomas, which we term were more ramified Tmem119+ microglia in the FF and
optic glioma stem cells (o-GSCs).15 For these studies, we FM1149C optic nerves (no tumors), whereas more amoe-
generated at least 3 independent o-GSC isolates from boid microglia were detected in the FMC, FMC, FM1278C,
3-month-old FMC, FMPC, FM1149C, and FM1278C mice and FMPC optic nerves bearing gliomas.
and 6-month-old FMOC mice, which could be stably In contrast to microglia infiltration pattern, FMOC mice
propagated after 2 weeks in vitro (Fig. 1C). Each of these did not harbor more T cells in their optic gliomas, whereas
o-GSC populations expressed nestin, sox2, BLBP, and both FM1278C and FMPC optic gliomas had ~2.5-fold more
CD133, characteristic of NSCs (Fig. 1D).15 Surprisingly, T cells than FMC mice (Fig. 3C). The differences between
FMC o-GSCs exhibited the highest level of proliferation, these strains are striking, specifically in the FM1278C
as determined by neurosphere diameter, EdU incorpo- strain, which exhibits a dissociation between the numbers
ration, Ki67 immunodetection, and direct cell counting of microglia and T cells. To characterize these infiltrating T
(Fig. 2A–D). In addition, FMC o-GSCs showed the highest cells, we performed immunohistochemistry using CD4 and
level of self-renewal at limiting dilution (Fig. 2E). This is in CD8 antibodies. While no CD4+ T cells were found in the
striking contrast to the overall tumor proliferation patterns optic gliomas (data not shown), the majority of the T cells
observed in vivo, where FMPC tumors had the highest were CD8+ cells (Fig. 3D). When examined at the level of
level of glioma proliferation. the individual tumor, nearly all CD3+ cells were also CD8+,
Since FMPC optic gliomas had much higher levels of with only a minor population of CD3+/CD8neg cells.
tumor proliferation relative to their FMC counterparts in Given our recent finding that T cells can “educate”
situ, we examined o-GSC survival in vitro. Leveraging prior microglia to produce Ccl5, a key growth factor required
RNA sequencing data from FMC and FMPC o-GSCs (Gene for optic glioma formation and growth,21 we sought to de-
Expression Omnibus accession number, GSE102345),18 termine whether Ccl5 levels correlated with proliferation
increased Bax, Bok, and Lrdd expression was detected in these tumors. RNA was extracted from the optic nerves
in FMPC o-GSCs, which was validated by real-time qRT- of 3-month-old FF, FMC, FMPC, and FM1278C mice (n = 4).
PCR on freshly isolated o-GSCs (Supplementary Fig. 2A, We specifically focused on these strains, in light of their
B). Consistent with increased Bax, Bok, and Lrdd expres- differences in T cell and microglia content. While the optic
sion, FMPC o-GSCs had more cleaved caspase-3 expres- gliomas from all three strains (FMC, FM1278C, and FMPC)
sion (Fig. 2F) and percentage of cells positive for terminal exhibited higher Ccl5 expression relative to FF mice (Fig. 4A,
deoxynucleotidyl transferase deoxyuridine triphosphate B), the differences in Ccl5 gene expression correlated with
nick end labeling (TUNEL) (Fig. 2G) relative to FMC o-GSCs the levels of optic nerve proliferation (%Ki67+ cells, R2 = 0.97;
following 48 h of growth factor starvation. Fig. 4C). For these studies, we coupled Ccl5 levels by qRT-
Lastly, the NF1 protein (neurofibromin) is a nega- PCR analysis (Fig. 4A) with immunohistochemistry (Fig. 4B;
tive Ras regulator, such that o-GSCs harboring biallelic %Ccl5+ cells), with identical results. Since Ccl5 levels are
Nf1 gene inactivation should have higher levels of Ras controlled by T cell interactions with microglia, we analyzed
activation. However, comparisons of the levels of Ras microglia (Fig. 4D) and T cell (Fig. 4E) content in each strain,
or Ras effector (extracellular signal-regulated kinase) and generated an “immune cell index,” representing the
activation did not reveal differences between the var- sum of the fold increases in T  cell and microglia content
ious o-GSC populations that could account for the relative to FF control mice (Fig. 4F). While neither T  cell
observed differences in o-GSC proliferation or apoptosis nor microglia content alone explained the Ccl5 levels, the
(Supplementary Fig. 2C, D). Taken together, these unex- combination of both (immune index) correlated with both
pected results indicate that the cell autonomous growth optic nerve Ccl5 levels (Fig. 4G) and proliferation (Fig. 4H).
properties of the neoplastic cells did not explain the FMOC optic gliomas also exhibited higher Ccl5 levels rel-
differences in tumor proliferation seen in situ. ative to normal FF optic nerves, whereas the optic nerves
Next, we evaluated the hypothesis that the biological from FM1149C mice, which do not form gliomas with sig-
heterogeneity observed in the intact tumors reflected nificant penetrance, did not exhibit higher Ccl5 expression
differential recruitment of immune system–like cells (Supplementary Fig. 3).
 Guo et al. Stromal regulation of low-grade glioma growth 5

Oncology
Neuro-
  
A
Additional
Cell of Germline Nf1 Age of Glioma

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Mouse strain Name genetic
origin mutation formation
mutation
Nf1flox/flox FF None No tumor

Nf1flox/neo; Neural
FMC Neo KO None 3mo
GFAP-Cre stem cell
Nf1flox/neo;
Neural Heterozygous
Ptenflox/WT FMPC Neo KO 3mo
stem cell Pten deletion
GFAP-Cre
Nf1flox/neo; Progenitor
FMOC Neo KO None 6mo
Olig2-Cre cell
flox/1149
Nf1 ; Neural 25% tumor
GFAP-Cre FM1149C Cys383* None penetrance
stem cell
Nf1flox/1278; Neural
FM1278C Arg1278Pro None 3mo
GFAP-Cre stem cell

B
12 ***
9
3
% Ki67+ cells

* *
2 *
N.T.

0
FF

PC
FM

49

78

FM
FM
11

12
FM

FM

Nf1 GEM Optic glioma o-GSCs 1 week o-GSCs 2 week

after isolation after isolation

D
FMC FMC1149C FM1278C FMOC FMPC

Nestin

Sox2

BLBP

CD133

Fig. 1  Isolation of o-GSCs from the Nf1 optic glioma mouse strains. (A) Summary of the Nf1 GEM strains used in this study, which differ by the
germline Nf1 gene mutation, cell of origin, or additional genetic alteration. (B) Nf1 optic glioma strains exhibit differences in overall tumor prolif-
eration (%Ki67+ cells). (C) Schematic of the workflow used to isolate o-GSCs from the various Nf1 murine optic glioma strains. Asterisks indicate
the location of tumor. (D) Stem cell markers (nestin, sox2, BLBP, and CD133) were expressed in the o-GSCs isolated from all of the Nf1 murine
optic glioma strains examined. Data are presented as mean ± SEM. *P < 0.05, ∗∗∗P < 0.001; N.T. denotes the low penetrance of optic gliomas in the
FM1149C strain. Scale bar, 40 μm.
6 Guo et al. Stromal regulation of low-grade glioma growth

  
A ***
FMC FMC1149C FM1278C FMOC FMPC 250

200

Diameter (um)

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150

100

50

PC
FM

49

78

O
B

FM
FM
11

12
C

FM
FM
40 ***

% Edu cells
30

+
20

10

PC
FM

49

78

FM
FM
11

12
C

FM
FM
40 ***

% Ki67 cells
30

+
20

10

PC
FM

49

78

FM
FM
11

12
D

FM
E

FM
20 50 ***
***
Neurospere number

40
15
×10 cells/well

30
10
20
4

5 10

0
0
C

PC
FM

49

78

O
C

PC

FM
FM
FM

49

78

11

12
FM
FM
11

12

FM
FM
C

FM
FM

F FMC FMPC
1 2 3 1 2 3
4
α-tubulin
c-caspase 3/caspase 3

*
3
caspase 3
2

0
c-caspase 3
C

PC
FM

FM

G FMC FMPC
1 2 3 1 2 3
8

TUNEL 6
% TUNEL cells
+

2
Merge
0
C

PC
FM

FM

Fig. 2  Optic GSCs exhibit different levels of proliferation, self-renewal, and apoptosis. Optic GSC neurosphere diameters (A), %EdU+ cells (B),
%Ki67+ cells (C), and direct cell counting (D) demonstrate that FMC o-GSCs exhibit the highest level of proliferation, as well as the highest levels
of self-renewal. (E, F) Immunoblotting revealed more cleaved caspase-3 in FMPC o-GSCs after 48 hours starvation relative to FMC o-GSCs. (G)
TUNEL assay revealed that FMPC o-GSCs have a higher percentage of TUNEL+ cells relative to FMC o-GSCs after 48 hours starvation. Data are
presented as mean ± SEM. *P < 0.05, ∗∗∗P < 0.001. Scale bar: 40 μm.

To determine the etiologic basis for these differences in Ccl2, and Ccl12 were detected in the CM from FMPC rela-
glioma T cell and microglia recruitment, we hypothesized tive to FMC o-GSCs (Fig. 5A, Supplementary Fig. 4A–C). In
that the neoplastic cells themselves might pro- addition, Ccl12 was increased, but Cx3cl1, Cxcl1, and Ccl2
duce chemokines, which recruit T cells and microglia. levels were reduced in FM1278C relative to FMC o-GSC CM.
Conditioned medium collected from FMC, FMPC, and Since these array experiments did not provide quantitative
FM1278C o-GSCs was assayed using a commercially avail- data, we measured the expression of these chemokines at
able chemokine array. Increased Cx3cl1, Cxcl6, Cxcl10, the mRNA level using qRT-PCR (Fig. 5B, Supplementary
 Guo et al. Stromal regulation of low-grade glioma growth 7

Oncology
Neuro-
  
A FF FMC FM1149C

24 ***

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20

% Ibal cells
10 ***
**

+
N.S.
FM1278C FMOC FMPC N.S.
5

FF

FM MC

PC
49

78

O
FM
FM
F
11

12
FM
B FF FMC FM1149C

25
***

% TMEM119 cells
20

+
15
* N.S. *
FM1278C FMOC FMPC 10 N.S.

FF

FM MC

PC
49

78

O
FM
FM
F
11

12
FM
C FF FMC FM1149C

20
** ***
15
CD3 cells

10
+

FM1278C FMOC FMPC *

5
N.S. N.S.
0
FF

PC
FM

49

78

O
FM
FM
11

12
FM

FM

FF FMC FM1149C
D
15
CD8 cells/optic nerve

***
10
**
FM1278C FMOC FMPC 5 * N.S.
+

N.S.
0
FF

PC
FM

49

78

O
FM
FM
11

12

40 um
FM

FM

Fig. 3  Differential infiltration of microglia and T cells in the Nf1 optic glioma strains. (A, B) FMC, FMOC, and FMPC mice exhibit more Iba1+
and Tmem119+ cells (microglia; %Iba1+ or %Tmem119+ cells) relative to FF mice, where FMPC optic gliomas have the highest levels of microglia
infiltration. (C) FMC, FM1278C, and FMPC mouse optic gliomas have greater CD3+ T  cell infiltration relative to FF mice, where FM1278C and
FMPC murine optic gliomas have the highest level of T cell infiltration. (D) The majority of the CD3+ cells are CD8+ T cells. Data are presented as
mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar: 40 µm.

Fig. 4D, E). Because only differential expression of Cx3cl1, highest Cx3cl1 (Cx3cr1 ligand) expression (FMC and FMPC)
Ccl2, and Ccl12 were validated (Fig. 5B, Supplementary attracted more microglia than CM  from FM1278C o-GSCs
Fig. 4D), these 3 chemokines were chosen for further study. (Fig. 5C). This observation also correlates to the microglia in-
First, we evaluated the expression of the receptors for filtration pattern in FMC and FMPC optic gliomas, relative to
these chemokines (Cxcl1-Cxcr2, Cx3cl1-Cx3cr1, Ccl2-Ccr2/ their FM1278C counterparts (Fig. 3A, B). The importance of
Ccr4, and Ccl12-Ccr2) in microglia using online RNA expres- Cx3cr1 to Nf1 optic glioma formation is further underscored
sion atlases (Supplementary Figures 5, 6).23,24 Consistent by the observation that FMC mice with reduced Cx3cr1 ex-
with earlier reports,25 only Cx3cr1 was highly expressed in pression have delayed optic glioma formation.20
microglia. The expression of Cx3cr1 on microglia is con- Second, to determine whether Ccl2 and Ccl12 were re-
sistent with our finding that the CM from 2 o-GSCs with the sponsible for T cell attraction, we examined the expression
8 Guo et al. Stromal regulation of low-grade glioma growth

  
A B
10
** 30
***
Relative Ccl5 level

FF FMC FM1278C FMPC

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8

ON %Ccl5+
6 20 *
*
4
* 10
2
40 um
0
0
FF

12 C
C
PC

FF

PC
FM FM
78
FM

FM

78

FM
12
FM
C D E F
8 25 *** 20 20
CD3+
Relative %Ki67 cells level

20 ** ** Ibal+

Microglia/T cells
6 15 15 15
15
% Ibal+ cells

CD3 cells
4 10 10
+

10

+
*
2 5 5 5
R2 = 0.97
0 0 0 0
2 4 6 8
FF

FM MC

C
PC

FF

FM MC

C
PC

FF

FM MC

C
PC
78

78

78
Ccl5 levels
FM

FM

FM
–2
F

F
12

12

12
G H
Relative %Ki67+ cells level

8 8

6 6
Ccl5 level

4 4

2 2
R2 = 0.97 R2 = 0.98
0 0
0 5 10 15 0 5 10 15
Relative microglia and T cell levels Relative microglia and T cell levels

Fig. 4  Microglia and T cell content correlates with Ccl5 levels and Nf1 optic glioma proliferation. (A) Quantitative RT-PCR revealed increased
optic glioma Ccl5 gene expression in FMPC (7-fold) and FM1278C (2.1-fold) mice relative to FMC mice. No Ccl5 expression was detected in FF
optic nerves. (B) FMC, FM1278C, and FMPC mice exhibited a greater percentage of Ccl5+ cells relative to FF mice by immunohistochemistry. Scale
bar: 40 μm. (C) Optic nerve Ccl5 expression correlates (R2 = 0.97) with relative tumor proliferation (relative %Ki67+ cells). Graphs demonstrating
the %Iba1+ cells (D), number of CD3+ cells (E), and the sums of the relative increases of microglia and T cells in the FMC, FM1278C, and FMPC optic
gliomas relative to normal (FF) optic nerve (F). The sum of the relative increases of microglia and T cells combined correlates (R2 = 0.97) with optic
nerve (G) Ccl5 expression and (H) proliferation (relative %Ki67+ cells). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01.

of their cognate receptors using the Immunological Ccl12 neutralizing antibodies together exhibited an addi-
Genome Project online database RNA expression atlas23 tive effect, suggesting that both chemokines are involved
(Supplementary Fig. 6). As expected, Ccr2 and Ccr4 are in T cell attraction by FMPC o-GSCs (Fig. 6E).
highly expressed in T cells. Consistent with a role for Ccr2
in Nf1 optic glioma T cell infiltration, the number of CD3+
cells was reduced in FMC mice heterozygous for a knockout
Ccr2 allele (Ccr2RFP/+ mice; Fig. 6A). Consistent with a crit- Discussion
ical role for Ccr2 engagement in the pathogenesis of Nf1
optic glioma, the percentages of Ki67+ cells and Ccl5+ cells One of the barriers to the implementation of precision on-
were reduced in FMC-Ccr2RFP/+ mice relative to their FMC cology is an incomplete elucidation of the molecular and
counterparts (Fig. 6B, C). Since FM1278C o-GSCs produce cellular etiologies that underlie tumor heterogeneity.26
mainly Ccl12, we sought to determine whether Ccl12 in- This is challenging using human tumor specimens, as the
hibition alone would attenuate T  cell migration using a variables that underlie differential tumor variability cannot
Transwell assay. Following exposure to Ccl12 neutralizing be easily controlled. Moreover, the experimental platforms
antibodies, T cell migration was reduced (Fig. 6D). that currently exist exclude critical components of tumor
Third, FMPC o-GSCs produce both Ccl2 and Ccl12, which biology, notably the tumor microenvironment, which
each could attract T cells. To determine whether these provides key paracrine support to the growing tumor.27,28
chemokines performed redundant functions, FMPC o-GSC To circumvent some of these obstacles, we have previ-
CM was added to T cells in a Transwell assay in the pres- ously deployed murine strains genetically engineered to
ence of Ccl2 and Ccl12 neutralizing antibodies. While Ccl2 differentially harbor distinct determinants that each could
and Ccl12 neutralizing antibodies each reduced T  cell mi- contribute to glioma growth. In the current study, we lev-
gration in response to FMPC CM, the addition of Ccl2 and eraged a small series of novel Nf1 mutant mouse strains,
 Guo et al. Stromal regulation of low-grade glioma growth 9

Oncology
Neuro-
  
A Cx3cl1 Ccl2 Ccl12
2.5 5 8

Relative protein expression

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2.0 4
6
1.5 3
4
1.0 2
2
0.5 1

0.0 0 0
C

PC

PC

PC
FM

78

FM

78

FM

78
FM

FM

FM
12

12

12
FM

FM

FM
B Cx3cl1 Ccl2 Ccl12
3 4 15
Relative mRNA expression

***
*** ***
3
2 10

2 **
1 5
1
*
***
0 0 0
C

PC

PC

PC
FM

78

FM

78

FM

78
FM

FM

FM
12

12

12
FM

FM

FM

C
1.5

*
Relative microglia

1.0

***
0.5

0.0
M

M
C

C
C

8C

PC
FM

FM
12
FM

Fig. 5  Differential o-GSC chemokine production dictates microglia and T cell infiltration. (A) FMC and FMPC o-GSC CM have the highest levels
of Cx3cl1 expression, FMPC the highest level of Ccl2 expression, and FM1278C and FMPC the highest level of Ccl12 expression. (B) Differential
Cx3cl1, Ccl2, and Ccl12 RNA expression was confirmed by qRT-PCR. (C) Greater microglia migration was observed with FMPC o-GSC CM rela-
tive to FMC or FM1278C o-GSC CM. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

incorporating some of these critical variables, to reveal to glioma growth have been previously revealed using both
that cancer stem cells create differences in tumor growth low-grade and high-grade astrocytoma mouse models.27,28
through the elaboration of specific chemokines that attract As such, the processes that control monocyte (microglia
T cells and microglia and create a supportive microenvi- and macrophage) tumor infiltration and activation include
ronment. These findings raise several important points the requirement for colony stimulating factor 1 receptor,29
germane to our understanding of low-grade brain tumors. CX3CL1 receptor (CX3CR1),20,30 and CCL5 receptor func-
First, the composite behavior of the tumor reflects syn- tion.16,21 To further demonstrate that o-GSC–produced
ergistic relationships between cancer and noncancer cells. chemokines determine the expression of stromal factors
These critical contributions of the tumor microenvironment that drive immune cell infiltration and optic glioma growth,
10 Guo et al. Stromal regulation of low-grade glioma growth

  
A
FMC FMC-Ccr2+/–
10

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8

CD3+ cells
6

2 *

B 10

%Ccl5+ cells
6

2 *

C
2.5
2.0

%Ki67+ cells
1.5 *
1.0
0.5
40 um 0.0

/+
C

FP
FM

2 R
cr
-C
C
D E
FM
FMC CM FMC CM
FM1278C CM FMPC CM
1.5 * 2.0
*

1.5 *
Relative T cells
Relative T cells

1.0 *

1.0 *

0.5
0.5

0.0 0.0
anti-Ccl2 – – + anti-Ccl2 – – + – +
anti-Ccl12 – – – + +

F
o-GSCs

T cells
Ccl2
2
Ccl1

Cx3
cl1

Microglia

Ccl5

Fig. 6  Optic glioma T cell infiltration in Nf1 GEM is mediated by Ccl2 and Ccl12. (A–C) FMC-Ccr2+/− mice have fewer T lymphocytes, Ccl5+ cells,
and Ki67+ cells in their optic nerves relative to those from FMC mice. Inset depicts a representative CD3+, Ccl5+, and Ki67+ cell. (D) Greater T cell
migration was observed with FM1278C relative to FMC CM, which was blocked with Ccl12 neutralizing antibodies. (E) Greater T cell migration was
observed with FMPC compared with FMC o-GSC CM, which was inhibited by the addition of neutralizing Ccl2 and Ccl12 antibodies. (F) Proposed
model for the differential stromal cell recruitment by o-GSCs. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01. Scale bar: 40 μm.
 Guo et al. Stromal regulation of low-grade glioma growth 11

Oncology
Neuro-
future in vivo experiments, such as those employing the leading to both increased T cell and microglia infiltration, as
inhibition of key chemokines (eg, Cx3cl1, Ccl12) as previ- well as higher levels of tumoral Ccl5 expression. Collectively,
ously performed using Ccl2 neutralizing antibodies,16 will these findings support the idea that additional mutations
be necessary. Coupling these experiments with genetic confer unique properties on cancer stem cells to facilitate the
strategies and tumor explant modeling will help to firmly establishment of a supportive microenvironment through dif-

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establish the requirements for this immune circuitry. ferential chemokine production and immune cell recruitment.
Moreover, other tumor–stroma circuit interactions in- Fourth, the cooperativity between T cells and microglia
volve versican activation of microglia31 and toll-like re- is highlighted by the finding that Ccl5 levels reflect the
ceptor engagement.32 In this regard, human PA tumor abundance of T cells and microglia. Recently, we showed
cell lines frequently undergo senescence in culture, that T cells produce little Ccl5, even following CD28/CD3
which may reflect the need for trophic factors produced activation, but are capable of stimulating microglia to pro-
by non-neoplastic cells, which are not currently incor- duce Ccl5 through the elaboration of paracrine signals.16
porated into these in vitro model systems.7,33 The es- In this manner, we envision that similar levels of resident
tablished network of supportive signals from the tumor microglia Ccl5 expression in the Arg1278 optic glioma
microenvironment, especially in the case of low-grade strain relative to the Nf1 knockout strain results from
gliomas, highlights the need to consider therapies that greaterT cell infiltration and microglia Ccl5 induction by the
target these co-dependencies. This is underscored by the larger number of coexisting T cells. Along those lines, the
findings in this report, which demonstrate that cell-intrinsic highest levels of tumor Ccl5 expression were observed in
differences in o-GSC growth in vitro do not account for the the Nf1 optic glioma strain harboring a heterozygous Pten
observed heterogeneity in optic glioma growth in vivo. deletion. These o-GSCs had the highest levels of chemo-
Instead, it is the combined effects of T cells and microglia kine expression, especially Cx3cl1, Ccl2, and Ccl12, which
infiltration by o-GSCs, leading to the differential Ccl5 pro- are likely responsible for attracting T cells and microglia
duction, which are ultimately responsible for dictating the and facilitating maximal microglia education and Ccl5
growth pattern of the tumor as a whole. production. The importance of T cells to microglia func-
Second, the impact of specific germline NF1 gene muta- tion and the establishment of a supportive tumor micro-
tion on the intrinsic tumor cell properties (apoptosis, prolif- environment is highlighted by studies in human tumors,
eration) appears to be limited, since all patient derived Nf1 where CD4+ T cell content positively correlated with tumor
gene mutations were similar with respect to o-GSC growth. grade.41–43 Studies are currently under way to define the
The notable exception was o-GSCs from genetically immunologic circuitry that attracts T cells and microglia,
engineered mice (GEM) strains harboring the artificial Nf1 physiologically activates T cells, and culminates in T cell–
knockout allele. It is not clear why o-GSCs from mice with mediated microglia reprogramming, Ccl5 production, and
this non-naturally occurring mutation exhibit higher levels enhanced glioma growth (Fig. 6F).
of proliferation and self-renewal. While this mutation affects Taken together, the deployment of a series of Nf1 GEM
the Ras GTPase activating protein domain (similar to the optic glioma strains with different germline Nf1 gene
Arg1278 mutation), this proliferative advantage is not due mutations, secondary genomic alterations, and cell of or-
to higher levels of Ras or mitogen-activated protein kinase igin provides an unprecedented opportunity to explore
kinase (MEK) activation. Preliminary experiments suggest the cellular and molecular etiologies for low-grade glioma
that these differences might result from variations in levels clinical diversity. The finding that these etiologies converge
of cyclic adenosine monophosphate (cAMP); however, the on cancer stem cell chemokine attraction of microglia and
mechanism(s) governing cAMP homeostasis in o-GSCs are T cells establishes the foundation for co-dependent glioma
currently not known. Studies are currently in progress to circuitry. In this regard, low-grade gliomas likely harbor mul-
expand these analyses to o-GSCs from additional Nf1 optic tiple vulnerabilities that reflect these stromal dependencies,
glioma mouse strains with different germline Nf1 gene which could be exploited to design more effective therapies.
mutations, as well as to explore other signaling pathways Finally, our ability to dissect the factors that create hetero-
activated in these neoplastic cells. Nonetheless, these geneity provides additional opportunities to understand the
findings underscore the importance of the tumor microen- molecular and cellular pathogenesis of these tumors, as
vironment to tumor proliferation involving non–cell auton- well as to potentially develop more personalized treatments
omous (stromal) drivers of glioma growth. for these common childhood gliomas.
Third, the impact of coexisting genomic alterations on the
non–cell autonomous properties of tumor growth provides
an additional mechanism to control stromal production of
growth factors and influence glioma biology. In this regard, Supplementary Material
studies using experimental models of high-grade glioma
have demonstrated that AXL regulates the glioblastoma mi- Supplementary data are available at Neuro-Oncology
croenvironment,34 enhancer of zeste homolog 2 controls online.
monocyte M1 phenotypes,35 and isocitrate dehydrogenase
modulates immune system infiltration.36 While the number
of additional genomic alterations in PA is small, mutations
in ATRX, ALT, KIAA1549:BRAF, PTEN, FGFR1, and H3-K27M Keywords
have been reported.4,37–40 As observed in o-GSCs from murine
Nf1 optic glioma harboring a heterozygous Pten mutation, a precision oncology | glioma stem cells | tumor microenvi-
different chemokine module accompanies Pten mutation, ronment | chemokine
12 Guo et al. Stromal regulation of low-grade glioma growth

11. Toonen JA, Anastasaki C, Smithson LJ, et al. NF1 germline mutation dif-

Funding ferentially dictates optic glioma formation and growth in neurofibroma-
tosis-1. Hum Mol Genet. 2016;25(9):1703–1713.
This work was funded by grants from the National Cancer 12. Gutmann  DH, Donahoe  J, Brown  T, James  CD, Perry  A. Loss of neu-
Institute (1-R01-CA214146-01) and the National Institute of rofibromatosis 1 (NF1) gene expression in NF1-associated pilocytic

Downloaded from https://academic.oup.com/neuro-oncology/advance-article-abstract/doi/10.1093/neuonc/noz080/5485427 by OCLC user on 18 July 2019

Neurological Disorders and Stroke (1-R35-NS07211-01) to D.H.G, astrocytomas. Neuropathol Appl Neurobiol. 2000;26(4):361–367.
as well as UL1-TR000448 (Hope Center Viral Vectors Core and 13. Yzaguirre AD, Padmanabhan A, de Groh ED, et al. Loss of neurofibromin
the Genome Technology Access Center). Y.P. was supported by a Ras-GAP activity enhances the formation of cardiac blood islands in mu-
fellowship grant from the James S. McDonnell Foundation. rine embryos. Elife. 2015;4:e07780.
14. Saederup N, Cardona AE, Croft K, et al. Selective chemokine receptor
usage by central nervous system myeloid cells in CCR2-red fluorescent
protein knock-in mice. PLoS One. 2010;5(10):e13693.
15. Chen YH, McGowan LD, Cimino PJ, et al. Mouse low-grade gliomas con-
Acknowledgments tain cancer stem cells with unique molecular and functional properties.
Cell Rep. 2015;10(11):1899–1912.
We appreciate the technical assistance of Ms Yu Ma. 
16. Pan  Y, Xiong  M, Chen  R, et  al. Athymic mice reveal a require-
ment for T-cell-microglia interactions in establishing a microenvi-
ronment supportive of Nf1 low-grade glioma growth. Genes Dev.
2018;32(7-8):491–496.
Conflict of interest statement. None declared. 17. Hegedus  B, Banerjee  D, Yeh  TH, et  al. Preclinical cancer therapy in
a mouse model of neurofibromatosis-1 optic glioma. Cancer Res.
2008;68(5):1520–1528.
18. Pan Y, Bush EC, Toonen JA, et al. Whole tumor RNA-sequencing and de-
Authorship statement. XG and DHG conceived the study. XG and convolution reveal a clinically-prognostic PTEN/PI3K-regulated glioma
YP performed the experiments. XG and DHG wrote the manu- transcriptional signature. Oncotarget. 2017;8(32):52474–52487.
script. DHG supervised the study. 19. Daginakatte  GC, Gutmann DH.  Neurofibromatosis-1 (Nf1) het-
erozygous brain microglia elaborate paracrine factors that pro-
mote Nf1-deficient astrocyte and glioma growth. Hum Mol Genet.
2007;16(9):1098–1112.
AQ1
20. Pong  WW, Higer  SB, Gianino  SM, Emnett  RJ, Gutmann  DH. Reduced
References microglial CX3CR1 expression delays neurofibromatosis-1 glioma forma-
tion. Ann Neurol. 2013;73(2):303–308.
21. Solga AC, Pong WW, Kim KY, et al. RNA sequencing of tumor-associated
1. Sadighi Z, Slopis J. Pilocytic astrocytoma: a disease with evolving mo- microglia reveals Ccl5 as a stromal chemokine critical for neurofibroma-
lecular heterogeneity. J Child Neurol. 2013;28(5):625–632. tosis-1 glioma growth. Neoplasia. 2015;17(10):776–788.
2. Rodriguez  FJ, Ligon  AH, Horkayne-Szakaly  I, et  al. BRAF duplications 22. Bennett  ML, Bennett  FC, Liddelow  SA, et  al. New tools for studying
and MAPK pathway activation are frequent in gliomas of the optic nerve microglia in the mouse and human CNS. Proc Natl Acad Sci U S A.
proper. J Neuropathol Exp Neurol. 2012;71(9):789–794. 2016;113(12):E1738–E1746.
3. Jones  DT, Kocialkowski  S, Liu  L, et  al. Tandem duplication producing 23. Zhang  Y, Chen  K, Sloan  SA, et  al. An RNA-sequencing transcriptome
a novel oncogenic BRAF fusion gene defines the majority of pilocytic and splicing database of glia, neurons, and vascular cells of the cerebral
astrocytomas. Cancer Res. 2008;68(21):8673–8677. cortex. J Neurosci. 2014;34(36):11929–11947.
4. Jones  DT, Hutter  B, Jäger  N, et  al; International Cancer Genome 24. Shay  T, Kang  J. Immunological genome project and systems immu-
Consortium PedBrain Tumor Project. Recurrent somatic alterations nology. Trends Immunol. 2013;34(12):602–609.
of FGFR1 and NTRK2 in pilocytic astrocytoma. Nat Genet. 25. Liang KJ, Lee JE, Wang YD, et al. Regulation of dynamic behavior of
2013;45(8):927–932. retinal microglia by CX3CR1 signaling. Invest Ophthalmol Vis Sci.
5. Listernick R, Charrow J, Greenwald M, Mets M. Natural history of optic 2009;50(9):4444–4451.
pathway tumors in children with neurofibromatosis type 1: a longitudinal 26. Seoane J, De Mattos-Arruda L. The challenge of intratumour heteroge-
study. J Pediatr. 1994;125(1):63–66. neity in precision medicine. J Intern Med. 2014;276(1):41–51.
6. Campen CJ, Gutmann DH. Optic pathway gliomas in neurofibromatosis 27. Hambardzumyan  D, Gutmann  DH, Kettenmann  H. The role of mi-
type 1. J Child Neurol. 2018;33(1):73–81. croglia and macrophages in glioma maintenance and progression. Nat
7. Raabe  EH, Lim  KS, Kim  JM, et  al. BRAF activation induces transfor- Neurosci. 2016;19(1):20–27.
mation and then senescence in human neural stem cells: a pilocytic 28. Quail DF, Joyce JA. The microenvironmental landscape of brain tumors.
astrocytoma model. Clin Cancer Res. 2011;17(11):3590–3599. Cancer Cell. 2017;31(3):326–341.
8. Bajenaru ML, Hernandez MR, Perry A, et al. Optic nerve glioma in mice 29. Pyonteck SM, Akkari L, Schuhmacher AJ, et al. CSF-1R inhibition alters
requires astrocyte Nf1 gene inactivation and Nf1 brain heterozygosity. macrophage polarization and blocks glioma progression. Nat Med.
Cancer Res. 2003;63(24):8573–8577. 2013;19(10):1264–1272.
9. Kaul A, Toonen JA, Gianino SM, Gutmann DH. The impact of coexisting 30. Feng X, Szulzewsky F, Yerevanian A, et al. Loss of CX3CR1 increases ac-
genetic mutations on murine optic glioma biology. Neuro Oncol. cumulation of inflammatory monocytes and promotes gliomagenesis.
2015;17(5):670–677. Oncotarget. 2015;6(17):15077–15094.
10. Solga AC, Toonen JA, Pan Y, et al. The cell of origin dictates the tem- 31. Hu F, Dzaye O, Hahn A, et al. Glioma-derived versican promotes tumor
poral course of neurofibromatosis-1 (Nf1) low-grade glioma formation. expansion via glioma-associated microglial/macrophages Toll-like re-
Oncotarget. 2017;8(29):47206–47215. ceptor 2 signaling. Neuro Oncol. 2015;17(2):200–210.
 Guo et al. Stromal regulation of low-grade glioma growth 13

Oncology
Neuro-
32. Vinnakota K, Hu F, Ku MC, et al. Toll-like receptor 2 mediates microglia/ 38. Orillac  C, Thomas  C, Dastagirzada  Y, et  al. Pilocytic astrocytoma and
brain macrophage MT1-MMP expression and glioma expansion. Neuro glioneuronal tumor with histone H3 K27M mutation. Acta Neuropathol
Oncol. 2013;15(11):1457–1468. Commun. 2016;4(1):84.
33. Jacob  K, Quang-Khuong  DA, Jones  DT, et  al. Genetic aberrations 39. Reinhardt A, Stichel D, Schrimpf D, et al. Anaplastic astrocytoma with
leading to MAPK pathway activation mediate oncogene-induced piloid features, a novel molecular class of IDH wildtype glioma with

Downloaded from https://academic.oup.com/neuro-oncology/advance-article-abstract/doi/10.1093/neuonc/noz080/5485427 by OCLC user on 18 July 2019

senescence in sporadic pilocytic astrocytomas. Clin Cancer Res. recurrent MAPK pathway, CDKN2A/B and ATRX alterations. Acta
2011;17(14):4650–4660. Neuropathol. 2018;136(2):273–291.
34. Sadahiro H, Kang KD, Gibson JT, et al. Activation of the receptor tyrosine 40. Becker  AP, Scapulatempo-Neto  C, Carloni  AC, et  al. KIAA1549: BRAF
kinase AXL regulates the immune microenvironment in glioblastoma. gene fusion and FGFR1 hotspot mutations are prognostic factors in
Cancer Res. 2018;78(11):3002–3013. pilocytic astrocytomas. J Neuropathol Exp Neurol. 2015;74(7):743–754.
35. Yin Y, Qiu S, Li X, Huang B, Xu Y, Peng Y. EZH2 suppression in glioblas- 41. Han  S, Zhang  C, Li  Q, et  al. Tumour-infiltrating CD4(+) and CD8(+)
toma shifts microglia toward M1 phenotype in tumor microenvironment. lymphocytes as predictors of clinical outcome in glioma. Br J Cancer.
J Neuroinflammation. 2017;14(1):220. 2014;110(10):2560–2568.
36. Amankulor  NM, Kim  Y, Arora  S, et  al. Mutant IDH1 regulates 42. Zhang W, Wu S, Guo K, Hu Z, Peng J, Li J. Correlation and clinical sig-
the tumor-associated immune system in gliomas. Genes Dev. nificance of LC3, CD68+ microglia, CD4+ T lymphocytes, and CD8+ T
2017;31(8):774–786. lymphocytes in gliomas. Clin Neurol Neurosurg. 2018;168:167–174.
37. Rodriguez  FJ, Brosnan-Cashman  JA, Allen  SJ, et  al. Alternative 43. Yang I, Han SJ, Sughrue ME, Tihan T, Parsa AT. Immune cell infiltrate
lengthening of telomeres, ATRX loss and H3-K27M mutations in differences in pilocytic astrocytoma and glioblastoma: evidence of dis-
histologically defined pilocytic astrocytoma with anaplasia. Brain tinct immunological microenvironments that reflect tumor biology. J
Pathol. 2019;29(1):126–140. Neurosurg. 2011;115(3):505–511.