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Colony-stimulating factor 1 receptor blockade inhibits tumor growth by altering the polarization of tumor-associated macrophages in hepatocellular carcinoma
Jian-Yang Ao1,2,3, Xiao-Dong Zhu1,2, Zong-Tao Chai1,2, Hao Cai1,2, Yuan-Yuan
Zhang1,2, Ke-Zhi Zhang4, Ling-Qun Kong5, Ning Zhang1,2, Bo-Gen Ye1,2, De-Ning
Ma1,2, and Hui-Chuan Sun1,2
1 Liver Cancer Institute and Zhongshan Hospital, Fudan University, Shanghai 200032,
China; 2 Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of
Education, Shanghai 200032, China; 3 Department of Hepatobiliary Surgery, the First
Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000,
China; 4 Department of Hepatobiliary Surgery, Taizhou People’s Hospital, Taizhou,
Jiangsu 225300, China; 5 Department of Hepatobiliary Surgery, Binzhou Medical
College Affiliated Hospital, Binzhou, Shandong 256610, China
Running title: CSF-1R blockade and macrophage polarization
Key words: Colony-stimulating factor 1 receptor, tumor associated macrophage,
macrophage polarization, colony stimulating factor 1, colony stimulating factor 2
Financial Support: National Natural Science Foundation of China NO. 81372655
(X.D. Zhu), National Natural Science Foundation of China NO. 81472224(H.C. Sun).
Correspondence to: Dr. Hui-Chuan Sun, Liver Cancer Institute and Zhongshan
Hospital, Fudan University, 180 Fenglin Road, Shanghai, 200032, China. E-mail:
[email protected]; Fax: +86-21-64037181
Note: J.Y. Ao, X.D. Zhu, Z.T. Chai and H.Cai contributed equally to this study.
Word Count (excluding reference):4681; Number of Figures: 6; Number of Tables: 0.
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Abstract
Colony-stimulating factor-1 (CSF-1) and its receptor, CSF-1R, regulate the differentiation and
function of macrophages and play an important role in macrophage infiltration in the context of
hepatocellular carcinoma (HCC). The therapeutic effects of CSF-1R blockade in HCC remain
unclear. In this study, we found that CSF-1R blockade by PLX3397, a competitive inhibitor
with high specificity for CSF-1R tyrosine kinase, significantly delayed tumor growth in
mouse models. PLX3397 inhibited the proliferation of macrophages in vitro, but intratumoral
macrophage infiltration was not decreased by PLX3397 in vivo. Gene expression profiling of
tumor-associated macrophages (TAMs) showed that TAMs from the PLX3397-treated tumors
were polarized toward an M1-like phenotype compared with those from vehicle-treated
tumors. In addition, PLX3397 treatment increased CD8+ T-cell infiltration, whereas CD4+
T-cell infiltration was decreased. Further study revealed that tumor cell–derived CSF-2
protected TAMs from being depleted by PLX3397. In conclusion, CSF-1R blockade delayed
tumor growth by shifting the polarization rather than the depletion of TAMs. CSF-1R blockade
warrants further investigation in the treatment of HCC.
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Introduction
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide
(1). Many cancers, including HCC, arise from sites of infection and chronic inflammation (2).
Inflammation has been recognized as having a role in tumor initiation as well as tumor progression
(3,4). A tumor microenvironment largely shaped by inflammatory cells is favorable for tumor
growth and progression (5). Tumor-associated macrophages (TAMs) are a major component of
inflammatory cellular infiltrates in tumors and play a pivotal role in tumor progression in
inflammation-related cancer (6-10). Macrophage activation in response to different agents has
long been recognized as M1 and M2 macrophages. Differential cytokine production is a key
feature of polarized macrophages(11). The M1 phenotype macrophages typically produce
interleukin (IL)-12b and IL-23, whereas M2 macrophages produce IL-10, chemokine (C-C motif)
ligand (CCL)17, and CCL22 (6,12). Our previous studies found that colony-stimulating factor 1
(CSF-1) expression and TAM density (CD68 or CSF-1 receptor, CSF-1R) in the adjacent liver
tissue are associated with patient survival after resection of HCC (13-15), suggesting that
CSF-1/CSF1R may play an important role in tumor progression and macrophage polarization in
HCC(16).
CSF-1 is a cytokine that controls the differentiation and function of macrophages via its receptor,
CSF-1R. Ligand binding activates the receptor kinase through a process of oligomerization and
transphosphorylation. Cellular changes occur in macrophages under different circumstances,
which contributes to the heterogeneity and variability of TAMs (17). This plasticity is a hallmark
of myeloid cells, particularly those of the monocyte–macrophage lineage (18). Activated forms of
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macrophages, known as M1 and M2, are linked to lineage-determining growth factors in T helper
1 (Th1) and T helper 2 (Th2) cells (19). CSF-1 and granulocyte–macrophage colony-stimulating
factor (GMCSF, or CSF-2) are often used to obtain mature macrophages, and they are also
involved in the polarization of anti-inflammatory/protumorigenic and
pro-inflammatory/antitumorigenic macrophages, respectively (11,20,21).
We previously found that depletion of TAMs enhanced the antitumor effect of sorafenib in liver
cancer models (22,23), and a clinical trial has been initiated in our hospital to test the safety of a
combination treatment using sorafenib with zoledronic acid (ClinicalTrials.gov identifier:
NCT01259193). Another approach is the deactivation of TAMs by a CSF-1R inhibitor, which was
studied in diffuse-type giant cell tumor (24) and glioblastoma multiforme (25). PLX3397, a
competitive inhibitor with high specificity for CSF-1R tyrosine kinase, has been tested in several
studies, with promising therapeutic results (26-33).
In the present study, we tested the effect of PLX3397 in HCC mouse models and found that
PLX3397 treatment delayed tumor growth. This delay was mediated by altering TAM polarization
rather than by causing TAM depletion.
Materials and Methods
Cell lines
Four human HCC cell lines (HepG2, MHCC97-H, MHCC97-L, and HCCLM3), one
non-transformed human hepatocyte cell line (L-02), and one mouse HCC cell line (Hepa1-6) were
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used in this study. MHCC97-H, MHCC97-L, and HCCLM3 were established at our institute
(34,35). HepG2 and Hepa1-6 were obtained from American Type Culture Collection (ATCC,
Rockville, MD). Human hepatic stellate cell line LX2 was obtained from Shanghai Advanced
Research Institute, Chinese Academy of Sciences. These cell lines were maintained in Dulbecco’s
modified Eagle’s medium (DMEM) (Gibco BRL, NY) supplemented with 10% (v/v) fetal bovine
serum (FBS) (Gibco BRL).
Human monocyte THP-1 cells obtained from ATCC were cultured in RPMI 1640 (Invitrogen,
Carlsbad, CA) containing 10% FBS and supplemented with 10 mM Hepes (Gibco BRL). THP-1
was differentiated into macrophages by 24-h incubation with 160 nM phorbol 12-myristate
13-acetate (PMA; Sigma, St. Louis, MO) followed by 24-h incubation in RPMI medium.
Macrophages were further polarized to M1 macrophages by incubation with 10 pg/ml of
lipopolysaccharide (LPS; Sigma) and 20 ng/ml of interferon (IFN)-γ (R&D Systems, MN) and are
referred to as M(LPS+IFN-γ) cells. M2 macrophages were obtained by incubation with 20 ng/ml
of interleukin (IL)-4 (R&D Systems) and are referred to as M(IL4) cells (36).
A human umbilical vein endothelial cell (HUVEC) line was purchased from Allcells
(Shanghai, China) and was grown in Allcells completed medium supplemented with 10%
FBS. All cell lines obtained from the cell bank were tested for authentication using short tandem
repeat fingerprinting and were passaged for fewer than 6 months.
Isolation of TAMs and T cells from tumor tissues
Isolated TAMs were obtained from orthotopically implanted hepa1-6 tumors in C57BL/6 mice.
Specimens were minced with scissors and digested by incubation for 1 h at 37°C in high-glucose
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DMEM (Life Technologies, CA) containing 0.1% collagenase IV (Sigma). After being washed in
medium plus 10% FBS, the cell suspension was forced through a graded series of meshes to obtain
single-cell suspensions and centrifuged at 400 g for 30–40 minutes at 18–20°C on layers of
Lympholyte-M (Cedarlane Labs, Burlington, Ontario, Canada), after which the upper layer was
removed, thus leaving the lymphocyte layer undisturbed at the interface. TAMs were sorted using
Fluorescence-activated cell sorting (FACS, Becton Dickinson, Franklin Lakes, NJ) with CD45+,
CD11b+, and F4/80+ (37) (Becton Dickinson) according to the protocol listed at
http://www.immgen.org/Protocols/ImmGen%20Cell%20prep%20and%20sorting%20SOP.pdf .
TAMs growing in DMEM supplemented with 10% FBS were tested to determine their biological
characteristics.
CD8+ T and CD4+ T cells were purified using the CD8+ or CD4+ T Cell Isolation Kit from
Miltenyi Biotec, respectively. The purified T cell suspension was labeled with CFSE (Invitrogen).
1×105 CFSE labeled CD8+ or CD4+ T cells were added to CD3/CD28 coated plates (1.0 µg/ml
each, eBioscience) to induce T cell proliferation. Analysis of cells was performed on a BD
LSRFortessa cell analyzer after labeling with fluorescent antibodies and 7AAD (BD Bioscience)
to exclude dead cells.
ELISA
IFN-γ concentrations in the supernatants were measured by enzyme-linked immunosorbent assay
(ELISA) kit (R&D Systems) according to the manufacturer’s instruction. We collected the total
cell protein to assess the different cell numbers of the different groups. An equal volume of lysis
buffer was added before the total cellular protein extracted, and then bicinchoninic acid (BCA)
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assay was used to measure the protein concentration. Thereafter, the IFN-γ concentration was
normalized to the total cellular protein.
Flow cytometry analysis
Lymphocytes isolated from tumors were washed in PBS containing 0.5% (w/v) bovine serum
albumin (BSA). After cells were counted, single-cell suspensions in FACS buffer (1% IgG free
BSA in PBS; Jackson Immunoresearch, West Grove, PA) were incubated with 1 µl of Fc Block
(Becton Dickinson) for every 1 million cells for at least 15 min at 4°C. Surface staining was
performed in the dark for 30 min at 4°C in staining buffer. Cells were then incubated for 30 min at
4°C with the appropriate antibody or with a control in PBS containing 0.5% (w/v) BSA. Cells
were analyzed on a fluorescence activated cell sorter (BD LSRFortessa; Becton Dickinson).
Surface markers used for these experiments included F4/80 clone REA126 FITC (1:100, Miltenyi
Biotec, Germany), CD11b clone M1/70 PE-Cy7 (1:125, BD Pharmingen, NJ), Ly6G clone 1A8
AlexaFluor700 (1:125, BD Pharmingen), and MHC Class II clone M5/114.15.2 APC (1:100,
Miltenyi Biotec). Directly conjugated mouse immunoglobulin G1k was used for isotype controls.
Cells were then washed twice with staining buffer followed by fixation in 1% paraformaldehyde
(VWR, West Chester, PA). Cells were analyzed on a BD LSRFortessa cell analyzer and gating was
performed with FlowJo analysis software (TreeStar, Ashland, OR). Biexponential transformation
was adjusted manually when necessary.
Obtaining CSF1- and CSF2-stimulated macrophages from bone marrow–derived
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macrophages (BMDMs)
Femur and tibia bones were harvested from 8-week-old wild-type (WT) C57BL/6 mice. Bone
marrow was flushed out into cold PBS (Life Technologies) plus 2% heat-inactivated FBS, passed
through a needle five times to dissociate the cells, and then passed through a 70-μm cell strainer
(Becton Dickinson) to remove cell clumps, bone, hair, and other cells/tissues. After addition of
three volumes of NH4Cl solution (0.8% NH4Cl solution; Beyotime Institute of Biotechnology,
Jiangsu, China), the mixture was incubated on ice for 10 min to remove red blood cells; the cells
were then spun down and resuspended in cold PBS with 2% FBS (38). The harvested cells were
cultured in DMEM containing 10% FBS and supplemented with 10 ng/ml recombinant mouse
CSF1 (R&D Systems) or 10 ng/ml recombinant mouse CSF2 (R&D Systems) for 7 days to obtain
CSF1- or CSF2-induced BMDMs: M(CSF1) or M(CSF2) cells, respectively.
Immunohistochemical assay
Frozen sections (5 μm) of tumor samples were used to determine the cells with dual expression of
CD68 (1:250, AbD Serotec, CA; 1:200, Abcam, Cambridge, UK) and Fizz1 (1:100; Abcam), cells
with dual expression of CD68 and MHC-II (1:100; Abcam), or cells with dual expression of CD68
and CSF-1R (1:100; Genetex, TX) by immunofluorescent staining and were then imaged by
confocal laser scanning microscopy.
Macrophages growing on glass coverslips were fixed in 4% paraformaldehyde for 15 min, rinsed
three times with PBS for 5 min each time, and incubated in a protein-blocking solution for 30 min
at room temperature. After incubation with the primary antibody against CD68 and F4/80 (1:100;
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Abcam), CD68 and CSF-1R (1:100; Novus Biologicals, CO, USA), CD68 and CSF2Rα (1:100;
Abcam), or CD68 and CSF2Rβ (1:100; Abcam) overnight at 4°C followed by incubation with the
secondary antibody (Alexa Fluor 488 donkey anti-rat, Alexa Fluor 546 donkey anti-rabbit, 1:300;
Jackson Immunoresearch) at 37°C for 2 h, the cells were counterstained with DAPI (Beyotime).
Cells on slides not incubated with primary antibodies served as negative controls.
Immunohistochemical assay was performed on 6-μm sections of paraffin-embedded Hepa1-6
tumor tissues using antibodies against CD8 (1:100; Abcam) and CD4 (1:100; Abcam). For
observing the staining for each antibody, a uniform setting was applied for all slides. CD68- or
CSF-1R-positive areas in the photographs were measured by the Leica Qwin Plus, and the
macrophage density in each photograph was calculated as CD68-positive area/total area. The
positive staining area values were determined as described previously (13).
Quantitative real-time polymerase chain reaction analysis
Total RNA from L-02, HCC cell lines (MHCC97-H, MHCC97-L, HCCLM3, and HepG2) and
Hepa1-6, mouse M(CSF1), M(CSF2), M(LPS+IFN-γ), and M(IL4) cells was extracted and
reverse-transcribed onto single-stranded cDNA using the iScript cDNA synthesis kit (Bio-Rad
Laboratories, Hercules, CA). For quantitative real-time polymerase chain reaction (qPCR),
primers were designed by Sangon Biotech (Shanghai, China), and their efficiency was tested on a
genomic DNA dilution series. qPCR was performed with the Applied Biosystems 8100 HT
Sequence Detection System (Applied Biosystems, Carlsbad, CA). Expression of the
glyceraldehyde-3 phosphate dehydrogenase gene was used to normalize the expression of each
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gene. The primer sequences used to determine the expression of the target genes are listed in
Supplementary Tables S1 and S2.
Western blot analysis
Cells were lysed with RIPA Lysis Buffer (Santa Cruz Biotechnology, Dallas, TX) containing
protease inhibitors (Beyotime Institute of Biotechnology). The protein concentration was
determined using a bicinchoninic acid assay (Beyotime Institute of Biotechnology) and equalized
before loading. Aliquots of 25–50 μg of protein were separated by SDS-PAGE and transferred
onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). Membranes were blocked
and blotted with the relevant antibodies. Horseradish peroxidase–conjugated secondary antibodies
were detected with an enhanced chemiluminescence reagent (Beyotime). Antibodies against
phosphorylated CSF-1R (Cell Signaling Technology, CST, Danvers, MA), CSF-1R (CST), and
CSF2rβ (Abcam) were used to determine their expression. GAPDH was used as a loading control.
All antibody dilutions were 1:1000, except for the GAPDH antibody, which was used at a dilution
of 1:5000.
Cell proliferation assay
Aliquots of the cell suspension were inoculated into a 96-well plate (3 × 103 to 5 × 103 cells in 100
µl/well) and then incubated in a humidified incubator (37°C, 5% CO2). 10 µl of CCK-8 solution
(Dojindo Laboratories, Kumamoto, Japan) was added to each well, and the plate was the incubated
for 2–4 h (37°C, 5% CO2). Absorbance at 450 nm was measured by a microplate reader.
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Animal studies
Male C57BL/6 mice (6 weeks old) and male BALB/c mice (6 weeks old) were obtained from the
Shanghai Institute of Materia Medica, Chinese Academy of Science, and housed under specific
pathogen-free conditions. The experimental protocol was approved by the Shanghai Medical
Experimental Animal Care Commission.
Hepa1-6 cells (6 × 106 cells) in 200 μl of normal saline were implanted by subcutaneous injection
to obtain subcutaneous tumors. Twenty C57BL/6 mice were treated by orthotopic implantation of
1mm3 tumor into the liver from a subcutaneously growing one. Three days after implantation, the
mice were randomized into two PLX3397-treated groups (50 mg/kg/day, oral administration) or
two vehicle-treated groups. Each group included five mice. The mouse body weights were
measured every week. One PLX3397-treated group and one vehicle-treated group were
continuously observed for survival analysis. In the other two groups, tumors were resected after 5
weeks to obtain TAMs; the remaining tumor tissues were stored in 4% paraformaldehyde solution
for further study. Tumor volume was calculated according to the following formula: tumor volume
= (largest diameter × perpendicular height2)/2.
HepG2 cells (5 × 105 cells) or HCCLM3 (5 × 105 cells) in 200 μl of normal saline were implanted
by subcutaneous injection to obtain subcutaneous tumors. Nude mice were treated by orthotopic
implantation of 1mm3 tumor into the liver from a subcutaneously growing one. Twelve Balb/c
nude mice with orthotopic HepG2 or 10 Balb/c nude mice with orthotopic HCCLM3 tumors were
randomized into PLX3397-treated and vehicle-treated groups. Three days after implantation,
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PLX3397 50 mg/kg/day or vehicle solution was orally administered by gavage. Body weights
were measured every week. Tumors were removed after 5 weeks and stored in a 4%
paraformaldehyde solution.
Molecular characteristics of macrophages
THP-1 derived macrophages, including M(LPS+INFγ) and M(IL4) cells, were processed in Trizol
and then analyzed using the Affymetrix U133 Array platform. M(CSF1) cells, M(CSF2) cells, and
TAMs from mouse tumors were processed in Trizol within 4 h after sorting and then analyzed
using whole-mouse genome Affymetrix Mouse Gene 1.0 ST Arrays. Data files are available at the
GEO database https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE95407. The M1
macrophage-related genes for MHC II (HLA-DRA), TNF, IL1B, IL12B, IL23A, CD80, CD86,
IL6, and CXCL10 and the M2 macrophage-related genes for CCL17, MRC1, IL10, and IL4R
were used to characterize macrophage polarization (19). Hierarchical clustering analysis using the
aforementioned panel of genes was performed using MeV v4.6.0 software (http://www.tm4.org/;
TM4, Boston, MA). The matrix was presented graphically by coloring each gene expression on
the basis of a measured color range: lower limit “−6” was blue, upper limit “6” was red, and
midpoint value “0” was white. A hierarchical clustering algorithm usually required two main steps,
which were repeated to find the strains that were the most similar: the M1/M2-associated gene
result values from the same strain were assigned to its own cluster, and the two clusters that were
closest to each other were merged until only one large cluster resulted.
Reagents and antibodies
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Other reagents and antibodies used in this study are shown in Supplementary Table S3.
Statistical analysis
Statistical analysis was performed with SPSS for Windows (version 20.0; SPSS Inc., Chicago, IL).
Quantitative variables were analyzed by the independent samples t-test. Overall survival and time
to recurrence were assessed using the Kaplan–Meier method and compared with the log-rank test.
Statistical significance was defined by P<0.05. Western blots were analyzed with ImageJ
(National Institutes of Health, Bethesda, MD). Cell migration was analyzed by Image-Pro Plus as
described previously (39). Pearson correlation was used as a distance metric and the complete
linkage method was used in hierarchical clustering.
Results
Bone marrow–derived monocytes were polarized toward the M2-like or M1-like
phenotype by CSF1 or CSF2 stimulation
M(CSF1) cells (CSF1-induced BMDMs) had a spindle appearance with a colony growth pattern,
whereas M(CSF2) cells (CSF2-induced BMDMs) had a more rounded appearance with a pattern
of individual growth (Supplementary Fig. S1A, B). FACS assay showed that more than 95% of
M(CSF1) and M(CSF2) cells were positive for F4/80 (Supplementary Fig. S1).
Immunocytofluorescence assay further confirmed that M(CSF1) and M(CSF2) cells were positive
for the markers specific to macrophages, including CD68, F4/80, and CSF-1R (Fig. 1A, B).
CSF2Rα and CSF-2Rβ expression was found on M(CSF1) and M(CSF2) cells by
immunocytochemistry assay (Fig. 1C, D). However, BMDMs without CSF-1 stimulation were not
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positive for CD68, F4/80, and CSF-1R and were suspended in the culture medium.
To establish molecular signatures shared by human and mouse macrophages, we conducted a
whole-genome expression profiling assay (40,41), which identified differentially expressed genes
in M1-polarized macrophages (THP-1–derived macrophages stimulated by LPS+IFNγ) and
M2-polarized macrophages (stimulated by IL4). Of these, expression of M1-associated genes such
as HLA-DRA, TNF, IL1B, IL12B, IL23A, CD80, CD86, IL6, and CXCL10 was higher, while
expression of M2-associated genes such as CCL17, MRC1, IL10, and IL4RA was lower in
comparison with M(LPS+IFNγ) and M(IL4) cells, which is consistent with previous reports
(11,19,42).
Whole mouse genome expression profiling identified 1631 genes that were up-regulated and 2539
down-regulated genes in M(CSF2) cells compared with M(CSF1) cells. Of these, expression of
M1-polarized macrophage-related genes such as MHC-II, Tnf, Il1b, Il12b, Il23a, Cd80, Cd86, Il6,
and Cxcl10 was increased and expression of M2-polarized macrophage-related genes such as
Ccl17, Mrc1, Il10, and Il4ra was decreased in the M(CSF2) cells, compared with their
counterparts, suggesting that M(CSF2) cells were more likely to be M1-polarized macrophages,
whereas M(CSF1) cells were more likely to be M2-polarized macrophages. The expression of the
typical markers for M1- or M2-polarized macrophages was validated by qPCR to confirm the
gene expression levels (Supplementary Fig. S2A, B).
PLX3397 suppressed tumor growth without depletion of TAM infiltration in vivo
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In vitro, a population doubling assay showed that the IC50 of PLX3397(33) (Fig. 2A), a highly
selective CSF-1R inhibitor, for M(CSF1) cells was 22 nM (Fig. 2B), which is consistent with a
previous report (43). A similar inhibitory effect of exogenous CSF-1R antibody on M(CSF1) cells
was observed (Fig. 2C). Marked suppression of CSF-1R phosphorylation (p-CSF-1R) in M(CSF1)
cells could be achieved with PLX3397 (Fig. 2D). In contrast, the same doses of PLX3397 showed
no antiproliferation effect on Hepa1-6, MHCC97-H, HCCLM3, HepG2, HUVEC, T cells,
fibroblasts cells, and M(CSF2) cells (Supplementary Fig. S3A–G). In accordance with these
results, very low CSF-1R expression was detected in hepatocyte (L-02), hepatoma (MHCC97-H,
MHCC97-L, HCCLM3, Hepa1-6, and HepG2), or endothelial cell lines (HUVEC) as compared
with M(CSF1) and M(CSF2) cells (Supplementary Fig. S3H).
In an orthotopic C57BL/6 model with Hepa1-6 tumor cells, PLX3397 treatment suppressed tumor
growth by 70% without affecting mouse body weight compared to the vehicle-treated mice (Fig.
3A–C). PLX3397 treatment also prolonged survival of the tumor-bearing mice (median survival
time, 8.0 weeks versus 11.3 weeks; P = 0.026; Fig. 3D).
We next examined the key mechanism of the antitumor effects of PLX3397. The number of
CD68-positive or CSF-1R-positive macrophages in the tumors from the PLX3397-treated and
vehicle-treated mice were not statistically different (Fig. 4A, B). The number of cells with CD31
(P = 0.589) or α-SMA (P = 0.913) expression did not differ between the two groups
(Supplementary Fig. S4A–F), suggesting that PLX3397 may have no effect on the infiltration of
macrophages or tumor angiogenesis in vivo.
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Tumor-derived CSF-2 promoted TAM polarization toward an M1-like phenotype under treatment with PLX3397
As PLX3397 treatment did NOT deplete TAM infiltration in vivo, whether the phenotype of TAMs
changed or cytokines protect TAM from depletion in the microenvironment was further
investigated. To study whether the phenotype of TAMs was affected by PLX3397, we conducted
gene expression profiling on the isolated TAMs. We found 4342 up-regulated genes and 4570
down-regulated genes in the PLX3397-treated TAMs compared with the vehicle-treated TAM. The
expression profile in TAMs isolated from the PLX3397-treated tumors was similar to that of
M1-polarized macrophages, whereas the gene expression profile of TAMs from the vehicle-treated
tumors was similar to that of M2-polorized macrophages (Supplementary Fig. S5A,
https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE95407). The expression of the typical
markers for M1- or M2-polarized macrophages were validated by qPCR (Supplementary Fig.
S5B).
An immunocytofluorescence assay was conducted to examine the expression of Mrc1, MHC-II,
and CD68 in the PLX3397- or vehicle-treated tumors. The results showed increased expression of
MHC-II-positive macrophages and decreased expression of Mrc1-positive macrophages
(Supplementary Fig. S5C-D). Taken together, these results indicated that PLX3397 promoted a
shift in polarization of macrophages from M2 to M1.
Because TAMs from the PLX3397-treated tumors showed an M1-like phenotype, we hypothesized
that this transition was promoted by the cytokines secreted in the tumor microenvironment. The
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isolated macrophages from the PLX3397- and vehicle-treated tumors were not different in
proliferation (Fig. 5A). The remaining non-macrophage cells from the tumor tissue were cultured
for 48 h, and the conditioned medium (CM) was collected. The CM of non-macrophage cells from
PLX3397-treated mice, but not from the vehicle-treated mice, protected the M(CSF1) cells from
the antiproliferative effects of PLX3397 in vitro (Fig. 5B). These results indicated that some
cytokines from the intratumoral microenvironment protected TAMs from the antiproliferative
effects of PLX3397.
An antibody array (AAM-ANG-1; Raybiotech, Norcross, GA) was conducted to identify the
differentially expressed cytokines in the conditioned medium (CM) of non-macrophage cells from
PLX3397-treated and vehicle-treated tumors. The results showed that the expression of CSF2,
granulocyte colony-stimulating factor 3 (CSF3), IL3 (multi-CSF), and IFN-γ were higher in the
CM of non-macrophages from the PLX3397-treated mice than from the vehicle-treated mice (Fig.
5C,D). Furthermore, a population doubling assay showed that CSF2, IL3, and IFN-γ protected
M(CSF1) cells from the inhibitory effects of PLX3397. Among these cytokines, CSF2 was the
most potent (Fig. 5E–H). These results suggested that CSF2 signals could be an important survival
factor for macrophages under a CSF-1R blockade. In accordance with these results, PLX3397 did
not inhibit M(CSF2) cells in a population doubling assay at concentrations of 22 nM and 50,000
nM (Supplementary Fig. S6A); however, IC50 of PLX3397 for M(CSF1) cells in medium
containing 10 ng/ml CSF1 was 22 nM, whereas IC50 of PLX3397 for M(CSF1) cells in medium
containing 10 ng/ml CSF2 was 19,495 nM (Supplementary Fig. S6B, C), suggesting that CSF2
protected macrophages from PLX3397 treatment.
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PLX3397 treatment changed the intratumoral microenvironment
We also studied the effects of PLX3397 on immune cells in the mouse model by immunostaining
the antigen presenting macrophages (F4/80+MHC II+), myeloid-derived suppressor cells (MDSCs,
CD11b+Gr1+) cells as well as the CD8+ and CD4+ T cells. We found that the proportion of antigen
presenting macrophages was elevated (P = 0.041), while myeloid-derived suppressor cells were
decreased (P = 0.029) in the tumor tissues (Supplementary Fig. S7A,B). Furthermore, PLX3397
treatment increased the number of CD8+ cell (P = 0.023) and decreased the number of CD4+ cells
in tumors compared to the vehicle treatment (P = 0.002) (Supplementary Fig. S7C,D).
Furthermore, as mentioned above, we found that PLX3397 did not affect the proliferation of
fibroblasts and T cells directly in vitro (Supplementary Fig. S3 H, J-M).
PLX3397 treatment inhibited tumor growth in two xenograft models of HCC
In xenograft models derived from two human hepatoma cell lines, HepG2 and HCCLM3,
PLX3397 treatment suppressed tumor growth by 33% and 84%, respectively (P = 0.026 and P =
0.047, respectively; Supplementary Fig. S8A–D). In addition, there was no difference in CD68+
pan-macrophages, but there was an increased expression of M1-associated marker MHC-II and a
decreased expression of M2-associated marker MRC1 in the PLX3397-treated tumors compared
to the vehicle-treated tumors (Supplementary Fig. S9).
Discussion
The present study showed that PLX3397 treatment induced the transition of M2-polarized
macrophages to M1-polarized macrophages in tumors, which was mediated by inhibition of
CSF-1R on tumor-associated macrophages, delayed tumor growth, and prolonged survival of
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tumor-bearing mice.
Our findings support the idea that M(CSF2) cells are similar to M1-macrophages, whereas
M(CSF1) cells are similar to M2-macrophages, which is consistent with a previous report (25).
Other authors have indicated that significant differences in transcriptome level remained in
M(CSF1) cells and M2 macrophages (19,21,44); however, the transcriptome data from the present
study showed that the gene expression signature determined by a panel of genes in M(CSF1) cells
was similar to that of M2 macrophages, and that M(CSF2) cells was similar to M1 macrophages.
Using the same signature, we showed that TAMs from the PLX3397-treated tumors were similar
to the M(CSF2) cells and TAMs from the untreated tumor were similar to the M(CSF1) cells. We
also showed that the M2-polarized macrophages were transformed to M1-polarized macrophages.
These results are in agreement with previous data (40,41).
Several studies have suggested that CSF1 is the major chemoattractant in cancers, attracting TAMs
to the neoplastic microenvironment and differentiating them to protumorigenic types (13,45,46).
In the present study, we demonstrated that blockade of CSF-1R signaling in TAMs affects tumor
progression in multiple hepatoma models by polarization of TAMs toward the M1 phenotype in
animal hepatoma models. TAMs were not depleted, and they survived CSF-1R inhibitor treatment,
which was consistent with findings showing that M(CSF1) cells could survive in CSF2-containing
medium with CSF-1R inhibition. The cell shape and sensitivity to the CSF-1R inhibitor also
changed in line with adding CSF2 to the medium. One interesting finding was that expression of
CSF2, IL3, and IFN-γ in the tumor microenvironment was increased when PLX3397 treatment
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was applied to tumors. In the present study, we did not attempt to identify the source of CSF2 in
the tumor microenvironment. Other authors have reported similar findings (25,47) supporting
other survival factors that were increased when the tumor was treated with CSF-1R inhibitors. The
authors suggested that tumor cells might be a source of CSF2 that was affected by CSF-1R
inhibitor treatment. Notably, Swierczak et al. have reported that inhibition of CSF-1R/CSF-1
signaling by AFS-98 (a CSF-1R antibody) increased the granulocyte CSF (G-CSF; CSF3) level in
the serum of mice with breast tumors (47) and was associated with increased metastasis of
mammary tumors in the animal model. It was also reported that increased CSF2 promoted tumor
growth through paracrine action on stromal cells in skin carcinoma (48) and promoted epithelial–
mesenchymal transition and metastasis in breast cancer (49). Furthermore, Ries et al. reported
RG7155 (a CSF-1R antibody) depletes macrophages in vitro and in vivo (50). These discrepancies
may indicate that the CSF1/CSF-1R system may play different roles in different types of cancers.
A recent study demonstrated a collaborative interaction between macrophage and stromal cells
(fibroblasts) in forming a favorable environment for neuroblastoma development(51). Although
we have showed that fibroblasts were not directly affected by PLX3397 in the present study, it
would be interesting to explore if the effect of PLX3397 on tumor growth is partially mediated by
fibroblasts or other mesenchymal stromal cells(52), which may be affected by polarized
macrophages.
PLX3397 treatment results in more antigen presenting macrophages and CD8+ cells and fewer
MDSCs and CD4+ cells in tumors. These findings suggested antitumor immunity in the
microenvironment has been ameliorated by CSF-1R inhibition, which is consistent with depletion
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21
of TAMs come out with more activated CTLs (53).
In summary, we found that treatment with CSF-1R inhibitor PLX3397 delayed tumor growth in
both xenograft and allograft models, and this delay was probably mediated by the transition from
M2 macrophages to M1 macrophages in a TAM population induced by blockade of the
CSF1/CSF-1R signal pathway (Fig. 6). It would be interesting to investigate the effects of the
CSF-1R inhibitor on HCC patients because systemic treatment for HCC is not yet sufficient.
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Acknowledgments
We thank Gideon E. Bollag, PhD, and Parmveer Singh from Plexxikon Inc. (Berkeley, CA)
for supplying the PLX3397 compound and scientific expertise. This study was jointly
supported by grants from the National Natural Science Foundation of China (No. 81372655
and No. 81472224).
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Figure Legends
Figure 1. Expression of a series of macrophage markers were found in CSF1- and
CSF2-induced BMDMs. Confocal laser scanning microscopy images of CSF1-induced
BMDMs (M(CSF1) cells) and CSF2-induced BMDMs (M(CSF2) cells). F4/80 (green),
CSF-1R (green), and CD68 (red) were co-expressed in M(CSF1) cells (A) and M(CSF2) cells
(B). CSF2Rα (green), CSF2Rβ (green), and CD68 (red) were also co-expressed in M(CSF1)
cells (C) and M(CSF2) cells (D). Scale bar = 100 μm.
Figure 2. CSF-1R blockade led to CSF1-induced BMDM (M(CSF1) cells) death by
phosphorylated CSF-1R inhibition.
(A) Chemical structure of PLX3397. PLX3397 is an oral, potent receptor tyrosine kinase
inhibitor of CSF-1R. Molecular Weight: 417.81. Chemical Formula: C20H15ClF3N5. (B) A
population doubling assay showed that IC50 concentration of PLX3397 in M(CSF1) cells was
22 nM. (C) Culture medium with different conditions were performed to determine M(CSF1)
survival. A neutralizing antibody against CSF-1R (10 μg/ml, GeneTex, AFS98) led to
M(CSF1) cell death in DMEM containing 10% FBS and supplemented with 10 ng/ml
recombinant mouse CSF1 in a similar manner with 22 nM, 220 nM PLX3397 and the culture
medium without CSF1, compare with M(CSF1) culture in DMEM containing 10% FBS and
supplemented with 10 ng/ml CSF1, as determined by CCK8 assays (n = 3 replicates, *,
P<0.01). (D) Western blot analysis of M(CSF1) cells, which were cultured in medium without
CSF-1 for 12 h before stimulation then followed CSF-1 addition for the time points indicated
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(1.5, 3, and 5 min). A progressive increase of phosphorylated CSF-1R expression resulted,
which was effectively inhibited by 22 nM PLX3397; CSF-1R expression was not changed. In
lane 1, marked by #, M(CSF1) cells were continuously cultured with CSF-1. GAPDH was
used as a loading control.
Figure 3. CSF-1R inhibition restricted tumor growth and prolonged mouse survival in
an allograft C57BL/6 mouse model with orthotopic implanted hepa1-6 cells. (A) After
5-week treatment with PLX3397 or vehicle, the mice were killed and the liver tissue was
obtained. (B) The mean tumor volume in the PLX3397-treated group was significantly lower
than in the vehicle-treated group (P = 0.045). (C) Mice treated with PLX3397 had a similar
body weight compared with those in the control group at 5 weeks (P = 0.712). (D) The
survival times were compared between the mice continuously treated with PLX3397 and those
that received the vehicle,. The cumulative survival plots showed that PLX3397 significantly
prolonged the survival time of the tumor-bearing mice (P = 0.026). The mice of treated PLX3397
and vehicle group were die through cancer cachexia and no lung, peritoneal cavity metastasis was
found.
Figure 4. PLX3397 suppressed tumor growth without depletion of TAM infiltration but
promoted TAM polarization toward an M1-like phenotype. In the allograft mouse models,
the densities of CD68- and CSF-1R-positive cells in the tumors treated with PLX3397 or
vehicle were not significantly different (P = 0.915 and P = 0.842, respectively.) (A and B).
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29
Figure 5. Tumor-derived CSF-2 promoted TAM polarization toward an M1-like phenotype
under the treatment of PLX3397. (A) A population doubling assay found that TAMs from
PLX3397 and vehicle treatments had no difference in proliferation. (B) The remaining
non-macrophage cells from the tumor tissue were cultured for 48 h, and the conditioned
medium (CM) was collected. A population doubling assay found that PLX3397-treated
non-TAM CM could support M(CSF1) cell survival with 22 nM PLX3397, while vehicle
non-TAM CM could not support M(CSF1) cell survival with 22 nM PLX3397. (C, D)
Antibody array identified that CSF2, CSF3, IL3, and IFN-γ were higher in PLX3397-treated
non-TAM CM than the vehicle group. (E–H). A population doubling assay found that 10
ng/ml CSF2, 10 ng/ml IFN-γ, and 10 ng/ml IL-3 could support M(CSF1) cell survival with 22
nM PLX3397. In these protective factors for M(CSF1) cells treated with CSF-1R inhibitor,
CSF2 was the most potent among them.
Figure 6. A diagrammatic illustration summarized the findings of this study. Both CSF1
and CSF2 can support the cell differentiation of BMDMs. However, BMDMs stimulated by
the CSF1/CSF-1R pathway (M(CSF1) cells) showed an M2-like phenotype (alternative
activation), whereas BMDMs stimulated by CSF2/CSF2Rα and CSF2Rβ pathway (M(CSF2)
cells) showed an M1-like phenotype (classical activation). When the CSF1/CSF-1R signaling
pathway was blocked, the CSF2/CSF-2R signaling pathway alternatively dominated the
differentiation of M(CSF1) cells, and these cells showed an M1-like phenotype.
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Published OnlineFirst June 1, 2017.Mol Cancer Ther Jian-Yang Ao, Xiao-Dong Zhu, Zong-Tao Chai, et al. macrophages in hepatocellular carcinomagrowth by altering the polarization of tumor-associated Colony-stimulating factor 1 receptor blockade inhibits tumor
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