zhang , ke-zhi zhang , ling-qun kong , ning zhang , bo-gen ...€¦ · 1/6/2017 · jian-yang...

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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.

on December 2, 2020. © 2017 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-16-0866

http://mct.aacrjournals.org/

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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




20

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




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.




22

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).




23

References

1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA: a cancer journal for clinicians 2015;65(2):87-108 doi 10.3322/caac.21262.

2. Coussens LM, Werb Z. Inflammation and cancer. Nature 2002;420(6917):860-7 doi 10.1038/nature01322.

3. Wan S, Kuo N, Kryczek I, Zou W, Welling TH. Myeloid cells in hepatocellular carcinoma. Hepatology 2015;62(4):1304-12 doi 10.1002/hep.27867.

4. Wang L, Fu H, Nanayakkara G, Li Y, Shao Y, Johnson C, et al. Novel extracellular and nuclear caspase-1 and inflammasomes propagate inflammation and regulate gene expression: a comprehensive database mining study. Journal of hematology & oncology 2016;9(1):122 doi 10.1186/s13045-016-0351-5.

5. Balkwill F, Charles KA, Mantovani A. Smoldering and polarized inflammation in the initiation and promotion of malignant disease. Cancer cell 2005;7(3):211-7 doi 10.1016/j.ccr.2005.02.013.

6. Capece D, Fischietti M, Verzella D, Gaggiano A, Cicciarelli G, Tessitore A, et al. The inflammatory microenvironment in hepatocellular carcinoma: a pivotal role for tumor-associated macrophages. BioMed research international 2013;2013:187204 doi 10.1155/2013/187204.

7. Mantovani A, Germano G, Marchesi F, Locatelli M, Biswas SK. Cancer-promoting tumor-associated macrophages: new vistas and open questions. European journal of immunology 2011;41(9):2522-5 doi 10.1002/eji.201141894.

8. Allavena P, Mantovani A. Immunology in the clinic review series; focus on cancer: tumour-associated macrophages: undisputed stars of the inflammatory tumour microenvironment. Clinical and experimental immunology 2012;167(2):195-205 doi 10.1111/j.1365-2249.2011.04515.x.

9. Qian BZ, Pollard JW. Macrophage diversity enhances tumor progression and metastasis. Cell 2010;141(1):39-51 doi 10.1016/j.cell.2010.03.014.

10. Noy R, Pollard JW. Tumor-associated macrophages: from mechanisms to therapy. Immunity 2014;41(1):49-61 doi 10.1016/j.immuni.2014.06.010.

11. Mantovani A, Sozzani S, Locati M, Allavena P, Sica A. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends in immunology 2002;23(11):549-55.

12. Gordon S. Alternative activation of macrophages. Nature reviews Immunology 2003;3(1):23-35 doi 10.1038/nri978.

13. Zhu XD, Zhang JB, Zhuang PY, Zhu HG, Zhang W, Xiong YQ, et al. High expression of macrophage colony-stimulating factor in peritumoral liver tissue is associated with poor survival after curative resection of hepatocellular carcinoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2008;26(16):2707-16 doi 10.1200/JCO.2007.15.6521.

14. Jia JB, Wang WQ, Sun HC, Zhu XD, Liu L, Zhuang PY, et al. High expression of macrophage colony-stimulating factor-1 receptor in peritumoral liver tissue is associated with poor outcome in hepatocellular carcinoma after curative resection. The oncologist 2010;15(7):732-43 doi 10.1634/theoncologist.2009-0170.




24

15. Kong LQ, Zhu XD, Xu HX, Zhang JB, Lu L, Wang WQ, et al. The clinical significance of the CD163+ and CD68+ macrophages in patients with hepatocellular carcinoma. PloS one 2013;8(3):e59771 doi 10.1371/journal.pone.0059771.

16. Chai ZT, Zhu XD, Ao JY, Wang WQ, Gao DM, Kong J, et al. microRNA-26a suppresses recruitment of macrophages by down-regulating macrophage colony-stimulating factor expression through the PI3K/Akt pathway in hepatocellular carcinoma. Journal of hematology & oncology 2015;8:56 doi 10.1186/s13045-015-0150-4.

17. Laoui D, Van Overmeire E, De Baetselier P, Van Ginderachter JA, Raes G. Functional Relationship between Tumor-Associated Macrophages and Macrophage Colony-Stimulating Factor as Contributors to Cancer Progression. Frontiers in immunology 2014;5:489 doi 10.3389/fimmu.2014.00489.

18. Galdiero MR, Bonavita E, Barajon I, Garlanda C, Mantovani A, Jaillon S. Tumor associated macrophages and neutrophils in cancer. Immunobiology 2013;218(11):1402-10 doi 10.1016/j.imbio.2013.06.003.

19. Murray PJ, Allen JE, Biswas SK, Fisher EA, Gilroy DW, Goerdt S, et al. Macrophage activation and polarization: nomenclature and experimental guidelines. Immunity 2014;41(1):14-20 doi 10.1016/j.immuni.2014.06.008.

20. Verreck FA, de Boer T, Langenberg DM, Hoeve MA, Kramer M, Vaisberg E, et al. Human IL-23-producing type 1 macrophages promote but IL-10-producing type 2 macrophages subvert immunity to (myco)bacteria. Proceedings of the National Academy of Sciences of the United States of America 2004;101(13):4560-5 doi 10.1073/pnas.0400983101.

21. Joshi S, Singh AR, Zulcic M, Bao L, Messer K, Ideker T, et al. Rac2 controls tumor growth, metastasis and M1-M2 macrophage differentiation in vivo. PloS one 2014;9(4):e95893 doi 10.1371/journal.pone.0095893.

22. Zhang W, Zhu XD, Sun HC, Xiong YQ, Zhuang PY, Xu HX, et al. Depletion of tumor-associated macrophages enhances the effect of sorafenib in metastatic liver cancer models by antimetastatic and antiangiogenic effects. Clin Cancer Res 2010;16(13):3420-30 doi 10.1158/1078-0432.ccr-09-2904.

23. Zhu XD, Sun HC, Xu HX, Kong LQ, Chai ZT, Lu L, et al. Antiangiogenic therapy promoted metastasis of hepatocellular carcinoma by suppressing host-derived interleukin-12b in mouse models. Angiogenesis 2013;16(4):809-20 doi 10.1007/s10456-013-9357-6.

24. Cassier PA, Gelderblom H, Stacchiotti S, Thomas D, Maki RG, Kroep JR, et al. Efficacy of imatinib mesylate for the treatment of locally advanced and/or metastatic tenosynovial giant cell tumor/pigmented villonodular synovitis. Cancer 2012;118(6):1649-55 doi 10.1002/cncr.26409.

25. Pyonteck SM, Akkari L, Schuhmacher AJ, Bowman RL, Sevenich L, Quail DF, et al. CSF-1R inhibition alters macrophage polarization and blocks glioma progression. Nature medicine 2013;19(10):1264-72 doi 10.1038/nm.3337.

26. Coniglio SJ, Eugenin E, Dobrenis K, Stanley ER, West BL, Symons MH, et al. Microglial stimulation of glioblastoma invasion involves epidermal growth factor receptor (EGFR) and colony stimulating factor 1 receptor (CSF-1R) signaling. Molecular medicine (Cambridge, Mass) 2012;18:519-27 doi 10.2119/molmed.2011.00217.

27. De I, Nikodemova M, Steffen MD, Sokn E, Maklakova VI, Watters JJ, et al. CSF1 overexpression has pleiotropic effects on microglia in vivo. Glia 2014;62(12):1955-67 doi 10.1002/glia.22717.




25

28. He Y, Rhodes SD, Chen S, Wu X, Yuan J, Yang X, et al. c-Fms signaling mediates neurofibromatosis Type-1 osteoclast gain-in-functions. PloS one 2012;7(11):e46900 doi 10.1371/journal.pone.0046900.

29. Mok S, Koya RC, Tsui C, Xu J, Robert L, Wu L, et al. Inhibition of CSF-1 receptor improves the antitumor efficacy of adoptive cell transfer immunotherapy. Cancer research 2014;74(1):153-61 doi 10.1158/0008-5472.can-13-1816.

30. Patwardhan PP, Surriga O, Beckman MJ, de Stanchina E, Dematteo RP, Tap WD, et al. Sustained inhibition of receptor tyrosine kinases and macrophage depletion by PLX3397 and rapamycin as a potential new approach for the treatment of MPNSTs. Clinical cancer research : an official journal of the American Association for Cancer Research 2014;20(12):3146-58 doi 10.1158/1078-0432.ccr-13-2576.

31. Prada CE, Jousma E, Rizvi TA, Wu J, Dunn RS, Mayes DA, et al. Neurofibroma-associated macrophages play roles in tumor growth and response to pharmacological inhibition. Acta neuropathologica 2013;125(1):159-68 doi 10.1007/s00401-012-1056-7.

32. Sluijter M, van der Sluis TC, van der Velden PA, Versluis M, West BL, van der Burg SH, et al. Inhibition of CSF-1R supports T-cell mediated melanoma therapy. PloS one 2014;9(8):e104230 doi 10.1371/journal.pone.0104230.

33. Tap WD, Wainberg ZA, Anthony SP, Ibrahim PN, Zhang C, Healey JH, et al. Structure-Guided Blockade of CSF1R Kinase in Tenosynovial Giant-Cell Tumor. The New England journal of medicine 2015;373(5):428-37 doi 10.1056/NEJMoa1411366.

34. Li Y, Tang Y, Ye L, Liu B, Liu K, Chen J, et al. Establishment of a hepatocellular carcinoma cell line with unique metastatic characteristics through in vivo selection and screening for metastasis-related genes through cDNA microarray. J Cancer Res Clin Oncol 2003;129(1):43-51 doi 10.1007/s00432-002-0396-4.

35. Li Y, Tian B, Yang J, Zhao L, Wu X, Ye SL, et al. Stepwise metastatic human hepatocellular carcinoma cell model system with multiple metastatic potentials established through consecutive in vivo selection and studies on metastatic characteristics. J Cancer Res Clin Oncol 2004;130(8):460-8 doi 10.1007/s00432-004-0564-9.

36. Genin M, Clement F, Fattaccioli A, Raes M, Michiels C. M1 and M2 macrophages derived from THP-1 cells differentially modulate the response of cancer cells to etoposide. BMC Cancer 2015;15:577 doi 10.1186/s12885-015-1546-9.

37. Bain CC, Bravo-Blas A, Scott CL, Gomez Perdiguero E, Geissmann F, Henri S, et al. Constant replenishment from circulating monocytes maintains the macrophage pool in the intestine of adult mice. Nat Immunol 2014;15(10):929-37 doi 10.1038/ni.2967.

38. Ying W, Cheruku PS, Bazer FW, Safe SH, Zhou B. Investigation of macrophage polarization using bone marrow derived macrophages. Journal of visualized experiments : JoVE 2013(76) doi 10.3791/50323.

39. Chai ZT, Kong J, Zhu XD, Zhang YY, Lu L, Zhou JM, et al. MicroRNA-26a inhibits angiogenesis by down-regulating VEGFA through the PIK3C2alpha/Akt/HIF-1alpha pathway in hepatocellular carcinoma. PloS one 2013;8(10):e77957 doi 10.1371/journal.pone.0077957.

40. Martinez FO, Gordon S, Locati M, Mantovani A. Transcriptional profiling of the human monocyte-to-macrophage differentiation and polarization: new molecules and patterns of gene expression. Journal of immunology (Baltimore, Md : 1950) 2006;177(10):7303-11.

41. Martinez FO, Helming L, Milde R, Varin A, Melgert BN, Draijer C, et al. Genetic programs




26

expressed in resting and IL-4 alternatively activated mouse and human macrophages: similarities and differences. Blood 2013;121(9):e57-69 doi 10.1182/blood-2012-06-436212.

42. Vogel DY, Vereyken EJ, Glim JE, Heijnen PD, Moeton M, van der Valk P, et al. Macrophages in inflammatory multiple sclerosis lesions have an intermediate activation status. J Neuroinflammation 2013;10:35 doi 10.1186/1742-2094-10-35.

43. DeNardo DG, Brennan DJ, Rexhepaj E, Ruffell B, Shiao SL, Madden SF, et al. Leukocyte complexity predicts breast cancer survival and functionally regulates response to chemotherapy. Cancer discovery 2011;1(1):54-67 doi 10.1158/2159-8274.cd-10-0028.

44. Fleetwood AJ, Dinh H, Cook AD, Hertzog PJ, Hamilton JA. GM-CSF- and M-CSF-dependent macrophage phenotypes display differential dependence on type I interferon signaling. J Leukoc Biol 2009;86(2):411-21 doi 10.1189/jlb.1108702.

45. Goswami S, Sahai E, Wyckoff JB, Cammer M, Cox D, Pixley FJ, et al. Macrophages promote the invasion of breast carcinoma cells via a colony-stimulating factor-1/epidermal growth factor paracrine loop. Cancer research 2005;65(12):5278-83 doi 10.1158/0008-5472.can-04-1853.

46. Wyckoff J, Wang W, Lin EY, Wang Y, Pixley F, Stanley ER, et al. A paracrine loop between tumor cells and macrophages is required for tumor cell migration in mammary tumors. Cancer research 2004;64(19):7022-9 doi 10.1158/0008-5472.can-04-1449.

47. Swierczak A, Cook AD, Lenzo JC, Restall CM, Doherty JP, Anderson RL, et al. The promotion of breast cancer metastasis caused by inhibition of CSF-1R/CSF-1 signaling is blocked by targeting the G-CSF receptor. Cancer immunology research 2014;2(8):765-76 doi 10.1158/2326-6066.CIR-13-0190.

48. Mueller MM, Peter W, Mappes M, Huelsen A, Steinbauer H, Boukamp P, et al. Tumor progression of skin carcinoma cells in vivo promoted by clonal selection, mutagenesis, and autocrine growth regulation by granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. The American journal of pathology 2001;159(4):1567-79 doi 10.1016/s0002-9440(10)62541-2.

49. Su S, Liu Q, Chen J, Chen J, Chen F, He C, et al. A positive feedback loop between mesenchymal-like cancer cells and macrophages is essential to breast cancer metastasis. Cancer cell 2014;25(5):605-20 doi 10.1016/j.ccr.2014.03.021.

50. Ries CH, Cannarile MA, Hoves S, Benz J, Wartha K, Runza V, et al. Targeting tumor-associated macrophages with anti-CSF-1R antibody reveals a strategy for cancer therapy. Cancer cell 2014;25(6):846-59 doi 10.1016/j.ccr.2014.05.016.

51. Hashimoto O, Yoshida M, Koma Y, Yanai T, Hasegawa D, Kosaka Y, et al. Collaboration of cancer-associated fibroblasts and tumour-associated macrophages for neuroblastoma development. The Journal of pathology 2016;240(2):211-23 doi 10.1002/path.4769.

52. Lou G, Song X, Yang F, Wu S, Wang J, Chen Z, et al. Exosomes derived from miR-122-modified adipose tissue-derived MSCs increase chemosensitivity of hepatocellular carcinoma. Journal of hematology & oncology 2015;8:122 doi 10.1186/s13045-015-0220-7.

53. Mitchem JB, Brennan DJ, Knolhoff BL, Belt BA, Zhu Y, Sanford DE, et al. Targeting tumor-infiltrating macrophages decreases tumor-initiating cells, relieves immunosuppression, and improves chemotherapeutic responses. Cancer research 2013;73(3):1128-41 doi 10.1158/0008-5472.can-12-2731.




27

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




28

(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).




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.




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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