Oncotarget, Vol. 7, No. 30

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Research Paper

Blockade of the chemokine receptor, CCR5, reduces the growthof orthotopically injected colon cancer cells via limiting cancerassociated fibroblast accumulation
Yamato Tanabe1, Soichiro Sasaki1, Naofumi Mukaida1, Tomohisa Baba1
1

Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, Kanazawa, Japan

Correspondence to: Tomohisa Baba, email: sergenti@staff.kanazawa-u.ac.jp
Keywords: colorectal cancer, CCL3, CCR5, cancer-associated fibroblast, maraviroc
Received: January 26, 2016     Accepted: June 07, 2016     Published: June 22, 2016

ABSTRACT
We previously demonstrated that cancer-associated fibroblasts (CAFs)
accumulate at tumor sites through the interaction between a chemokine, CCL3, and its
receptor, CCR5, in the late phase of colitis-associated colon carcinogenesis. Here we
examined the effect of a CCR5 antagonist, maraviroc, on tumor growth arising fromthe orthotopic injection of mouse or human colon cancer cell lines into the cecal wallby focusing on CAFs. Orthotopic injection of either cell line caused tumor formation
together with leukocyte infiltration and fibroblast accumulation. Concomitant oral
administration of maraviroc reduced tumor formation with few effects on leukocyte
infiltration. In contrast, maraviroc reduced the intratumor number of α-smoothmuscle actin-positive fibroblasts, which express epidermal growth factor, a crucial
growth factor for colon cancer cell growth. These observations suggest that maraviroc
or other CCR5 antagonists might act as novel anti-CRC drugs to dampen CAFs, anessential cell component for tumor progression.

promote cancer progression and metastasis by supplying
cancer cells with oxygen and nutrients, which are
indispensable for cancer cell growth [5]. As a consequence,an anti-vascular endothelial growth factor (VEGF)
antibody is used to supplement cytotoxic chemotherapy
for CRC [6]. However, the antibody can prolong OS ofpatients with metastatic CRC by only a few months [2];therefore, additional targets need to be identified for theestablishment of a therapy for advanced CRC.
Fibroblasts present in tumor tissues, designated ascancer-associated fibroblasts (CAFs), can provide cancer
cells with scaffolds and growth factors to promote cancer
cell growth [7]. For example, the contribution of CAFderived growth factors has been documented in a mousecolitis-associated CRC model [8, 9]. We further revealed
that in this model, a chemokine, CCL3, induces theaccumulation of fibroblasts that express CCR5, a specificreceptor for CCL3 [10]. Additionally, genetic deletion of
the Ccr5 gene or in vivo overexpression of a dominantnegative form of the CCR5 ligand, CCL5, attenuatedtumor formation in this model together with reduced
fibroblast accumulation. Furthermore, we observed that
CCR5-deficient mice exhibited slower tumor growththan wild-type mice when a syngeneic mouse CRC cell

INTRODUCTION
Colorectal cancer (CRC) is the third most commonly
diagnosed cancer (1.36 million cases) and the fourthhighest cause of cancer deaths (0.7 million) worldwide [1].
Approximately three quarters of patients newly diagnosed
with CRC exhibit localized disease that is amenable
to curative surgical resection, whereas the remaining
patients present with unresectable metastatic disease[2]. Furthermore, a substantial proportion of patients
experience disease recurrence or metastatic disease after
surgical resection. The prognosis of CRC patients withmetastasis and/or recurrence has been improved by theuse of various types of chemotherapeutic drugs including
5-fluorouracil, leucovorin, irinotecan, and oxaliplatin[2, 3]. However, the median overall survival (OS) still
remains about 2 years [2]. Thus, an additional treatment
modality is required for advanced CRC.
Accumulating evidence indicates the crucial
involvement of inflammation in cancer progression
and metastasis [3]. Inflammation is pathologicallycharacterized by the presence of inflammatory cell
infiltration, fibrosis ensuing from fibroblast accumulation,
and angiogenesis [4]. Angiogenesis is presumed towww.impactjournals.com/oncotarget

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(Figure 2D). These observations suggest that maraviroc
might act on other types of cells present in tumor tissues
rather than on the cancer cells themselves.

line was injected either subcutaneously or orthotopicallyinto the cecal wall [10]. Therefore, in this study weinvestigated the potential of a clinically utilized CCR5
antagonist, maraviroc, for the treatment of CRC in mousepre-clinical models.

Maraviroc suppresses fibroblast accumulation intumors arising from the orthotopic injection of amurine CRC cell line

RESULTSWe next evaluated the effects of maraviroc on noncancer cells present in tumor tissues, such as endothelial
cells, fibroblasts, and leukocytes. Immunohistochemical
analysis demonstrated that tumor tissues additionallycontained CD31-positive endothelial cells and type I
collagen- or α-SMA-positive fibroblasts. Furthermore,maraviroc significantly reduced type I collagen- andα-SMA-positive areas but not CD31-positive areas(Figure 2E and 2F). Infiltrating leukocytes werecomprised mainly of Ly6G-positive granulocytes andF4/80-positive macrophages with a small number ofCD3-positive lymphocytes (data not shown); however,maraviroc had few effects on these infiltrating leukocyte
percentages (Figure 2F). Although CCR5 was detected
in immunosuppressive cells [11, 12], maraviroc failed tochange the intratumoral percentages of myeloid-derived
suppressor cells (MDSCs) and regulatory T cells (Tregs),
two major immunosuppressive cell types (Figure 2G). On
the contrary, a double-color immunofluorescence analysis
demonstrated that maraviroc reduced CCR5-expressing
α-SMA-positive fibroblasts (Figure 2H). Theseobservations imply that maraviroc reduced tumor
formation mainly by acting on fibroblasts.

Orthotopic injection of a murine CRC cellline forms tumors in association with CCR5expressing fibroblasts and CCL3-expressing
granulocytes and macrophages
We previously demonstrated that tumor formation
was significantly reduced in CCR5-deficient micecompared with WT mice when colon 26 cells were
injected into the cecal wall [10]. These observations
imply the possible involvement of the CCR5 axis incolon tumor progression. Hence, in the current study
we inoculated colon 26 cells into the cecal wall of WT
mice in order to delineate the molecular and cellularmechanisms underlying this phenomenon in more detail.
Tumors were macroscopically evident 7 days after the
injection and grew progressively (Figure 1A and 1B). We
next determined the expression of CCR5 and its ligands
in the tumor sites by quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR) analysis. The
expression of Ccr5 and its ligands, Ccl3 and Ccl4, but not
Ccl5, were markedly increased in tumor sites 7 days after
the injection (Figure 1C). Intratumoral CCL3 and CCL4
contents were also significantly increased at the protein
level 15 days after the injection (Figure 1D). Furthermore,a double-color immunofluorescence analysis demonstrated
that CCR5 was detected mainly in α-smooth muscle actin(SMA)-positive fibroblasts (Figure 1E). Flow cytometricanalysis illustrated that CCL3 proteins were expressedin Ly6G-positive granulocytes and F4/80-positive
macrophages but not in T cell receptor β chain (TCRβ)positive T cells (Figure 1F).

Maraviroc suppresses the migration offibroblasts expressing epidermal growth factor(EGF)
Accumulating evidence indicates that CAFs
are a rich source of various growth factors including
hepatocyte growth factor (HGF), EGF, heparin-binding
epidermal growth factor-like growth factor (HB-EGF),
and epiregulin (EREG). Among these, only Egf mRNA
expression was reduced by maraviroc treatment intumor tissues (Figure 3A). In addition, a double-color
immunofluorescence analysis revealed that EGF proteins
were detected in α-SMA-positive cells and that maraviroc
treatment reduced EGF-expressing and α-SMA-positive
cells (Figure 3B). Furthermore, in vitro exposure to CCL3
enhanced cell migration and EGF expression in a mouse
fibroblast cell line; this enhancement was abrogated bymaraviroc treatment (Figure 3C and 3D). Although CCL3
marginally increased EGF expression, the effect of CCL3
on fibroblast migration was evident. These observations
suggest that maraviroc primarily blocked the CCR5induced intratumoral accumulation and subsequent
activation of fibroblasts expressing EGF, an essential
growth factor for CRC cells [13].

Maraviroc suppresses the tumor formation of amurine CRC cell line
The potential involvement of the CCR5 axis incolon tumor progression prompted us to investigate
the effects of a CCR5 antagonist, maraviroc, on colon
tumor progression induced by the injection of colon 26
cells into the cecal wall. Maraviroc treatment reduced
tumor sizes by approximately half (Figure 2A and 2B).
However, maraviroc treatment failed to induce massive
necrosis and apoptosis in tumor cells (Figure 2C).
Similar observations were obtained when maraviroc
administration was instituted beginning at 8 days after
the tumor injection (data not shown). Consistent withthese findings, maraviroc exhibited few effects on thein vitro cell proliferation of colon 26 cells up to 100 μM
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Figure 1: Colon carcinoma progression with CCL3-expressing resident stromal cell accumulation. A and B. A mousecolon adenocarcinoma cell line, colon 26, was injected into the cecal wall of WT mice as described in Materials and Methods. Tumors were
resected at the indicated time intervals from the injection. Representative macroscopic appearance of tumors is shown in A. Tumor volumes
were determined at the indicated time intervals. Mean and SD were calculated and are shown in B (n = 8). C. Expression of Ccr5 and its
ligands in tumor tissues. Total RNAs were extracted from the tumor tissues obtained at the indicated time intervals and subjected to qRTPCR to determine the mRNA expression of CCR5 and its ligands, CCL3, CCL4, and CCL5. Data represent the means and SEM (n = 5).
Statistical significance was evaluated using one-way ANOVA, followed by Dunnett’s test. *p<0.05; **p<0.01; ns, not significant. D. CCL3
and CCL4 protein expression in tumor tissues. Tumor tissues were obtained at the indicated time intervals and the levels of CCL3 and CCL4
protein in the tumor tissue extracts were quantified using ELISA. Data represent the means and SEM (n = 6). p values were calculatedusing Mann-Whitney’s U test. **p<0.01. E. Double-color immunofluorescence analysis of CCR5-expressing cells using the combination ofanti-CCR5 and anti-α-SMA antibodies. Representative results from 5 independent experiments are shown. The scale bar indicates 50 μm.
F. Flow cytometry data of CCL3 expression by Ly6G-positive granulocytes, F4/80-positive macrophages, and TCRβ-positive T cells
isolated from tumor tissues. Single cell suspensions were obtained from tumor tissues at 15 days after tumor inoculation and were subjected
to flow cytometric analysis to determine the proportion of CCL3-expressing cells. Red lines and gray-filled histograms indicate the CCL3
expression and isotype-matched control, respectively. Representative results from three independent experiments are shown.

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Figure 2: Effects of maraviroc on colon 26 cell tumor formation. A. and B. Maraviroc was orally administered to mice that hadreceived an injection of colon 26 cells into the cecal wall as described in Materials and Methods. Tumors were resected at day 15 after
the injection. Representative macroscopic appearances of tumors are shown in A. Tumor volumes were determined and each symbol in B
indicates the tumor volume of each mouse. The mean value was calculated for each group (n = 15) and is shown with a bar. p values were
calculated using Mann-Whitney’s U test. **p<0.01. C. Microscopic appearance of tumors. Tumors were resected at day 15 after the injection
and were subjected to hematoxylin and eosin staining. Insets indicate high power magnification. Representative results from 5 individual
animals are shown. The scale bar indicates 50 μm. D. Effects of maraviroc on the in vitro cell proliferation of colon 26 cells. In vitro cellproliferation was conducted in quadruplicate as described in Materials and Methods. The means and SD values were calculated and are
as shown. E. and F. Tumor tissues were obtained at day 15 and subjected to immunohistochemical analysis using anti-Ly6G, anti-F4/80,
anti-CD31, anti-type I collagen, or anti-α-SMA antibodies. Representative microscopic appearances of CAFs in tumor tissues are shown in
E. The scale bar indicates 50 μm. Positive areas were determined as described in Materials and Methods. The means and SD values were
calculated from 5 animals and are shown in F. Statistical significance was evaluated using one-way ANOVA, followed by Tukey-Kramer’s
posthoc test. *p<0.05; **p<0.01; ns, not significant. G. Immunosuppressive cell infiltration into inoculated tumors. Single cell suspensions
were obtained from tumor tissues at 15 days after tumor inoculation and subjected to flow cytometric analysis to determine the populationsof CD11b+Gr-1+ MDSCs and CD25+Foxp3+ Tregs among the CD45 cells. Each symbol indicates a separate mouse. The mean value wascalculated for each group (control: n = 8, Maraviroc: n = 7) and is shown with a bar. p values were calculated using Mann-Whitney’s U test.
ns, not significant. H. Double-color immunofluorescence analysis of CCR5-expressing cells using anti-CCR5 and anti-α-SMA antibodies.
Representative results from 5 independent experiments are shown. The scale bar indicates 50 µm.
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Maraviroc suppresses the tumor formation of ahuman CRC cell line

CRC cell line, KM12C, into the cecal wall. As for colon
26 cells, maraviroc treatment reduced xenotransplanttumor formation without inducing apparent necrosisin the tumor cells (Figure 4A and 4B). Consistent
with these findings, maraviroc failed to inhibit thein vitro cell proliferation of KM12C cells up to 100 μM

To generalize the results obtained in the murine
model, we next examined the effects of maraviroc ontumor formation induced by the injection of a human

Figure 3: EGF expression by CAFs. A. Total RNAs were extracted from tumor tissues obtained at day 15 after the injection andsubjected to qRT-PCR analysis to determine the expression of growth factors. The means and SD values were calculated from 5 animals andare as shown. p values were calculated using Mann-Whitney’s U test. *p<0.05; **p<0.01. B. Double-color immunofluorescence analysis of
EGF-expressing cell was conducted with anti-EGF and anti-α-SMA antibodies. Representative results from 5 independent experiments areshown. The scale bar indicates 50 μm. C. Migration of fibroblasts in response to CCL3. Each value represents the means and SEM (n = 5).
p values were calculated using Mann-Whitney’s U test. *p<0.05; **p<0.01. D. Effects of CCL3 on fibroblast EGF expression. NIH3T3
cells were treated with CCL3 in the presence or absence of maraviroc, as described in Materials and Methods. Total RNAs were extracted todetermine Egf mRNA levels using qRT-PCR. The means and SD values were calculated from 8 independent experiments and are as shown.
Statistical significance was evaluated using one-way ANOVA followed by Tukey-Kramer’s posthoc test. *p<0.05; ns, not significant.
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(Figure 4C), similarly to its observed lack of effect oncolon 26 cells.

(data not shown). Furthermore, maraviroc treatment
significantly attenuated mouse Egf mRNA expression(Figure 4E). Thus, maraviroc likely inhibits the tumor
formation induced by the injection of a human CRC cellline into the cecal wall mainly by acting on CAFs.

Maraviroc suppresses fibroblast accumulation intumors arising from the orthotopic injection of ahuman CRC cell line

DISCUSSION

We next examined the effects of maraviroc on thecell components in tumors arising from the injectionof KM12C into the cecal wall. Maraviroc reduced
intratumoral type I collagen- or α-SMA-positive
fibroblast areas with few effects on CD31-positive areas(Figure 4D) or on Ly6G-positive granulocyte and F4/80positive macrophage percentages in the tumor sites

Fibroblasts are a major cell component of the tumor
microenvironment in various types of solid tumors [7].
Under homeostatic conditions, fibroblasts can act as asentinel cell to restrict pre-malignant cell growth [14]. In
contrast, CAFs can also contribute to tumor progression
by providing cancer cells with growth factors [8, 9] andFigure 4: Effects of maraviroc on KM12C cell tumor formation. A. and B. Maraviroc was administered orally to mice that hadreceived an injection of KM12C cells into the cecal wall. Tumors were resected at day 28 after the injection to determine tumor volumes.
Each symbol indicates the tumor volume of an animal and the means of each group (n = 6) is indicated with a bar. p values were calculatedusing Mann-Whitney’s U test. **p<0.01. Representative microscopic appearance of hematoxylin and eosin-stained tumor tissues is shown
in B; the insets indicate high power magnification. The scale bar indicates 50 μm. C. Effects of maraviroc on the in vitro cell proliferation of
KM12C cells. In vitro cell proliferation was conducted in quadruplicate as described in Materials and Methods. The means and SD values
were calculated and are as shown. D. Tumor tissues were obtained at day 28 after the injection and subjected to immunohistochemical
analysis using anti-CD31, anti-type I collagen, or anti-α-SMA antibodies. Positive areas were determined. The means and SD values were
calculated from 5 animals and are as shown. p values were calculated using Mann-Whitney’s U test. **p<0.01; ns, not significant. E. Total
RNAs were extracted from tumor tissues obtained at day 28 after the injection and subjected to qRT-PCR analysis to detect Egf mRNA
expression. The means and SD values were calculated from 5 animals and are as shown. The data were analyzed statistically using MannWhitney’s U test. **p<0.01.

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Oncotargetcells remains elusive. Nevertheless, the lack of CCR5
expression in colon 26 or KM12C cells argues against thedirect actions of maraviroc on cancer cells in our present
preclinical models.
Another potential mechanism for the observed
maraviroc-mediated tumor reduction in our model
is suggested by the results from Ohshio et al., who
administered a clinically used anti-fibrotic agent, tranilast, totumor-bearing mice [35]. Several lines of evidence indicatethat tranilast mainly interferes with the transforming growth
factor (TGF)-β-mediated signaling pathway to inhibit
fibrosis [36]. Tranilast was shown to significantly reduce
CAFs but had only marginal effects on in vivo tumor growth[35]. Conversely, maraviroc treatment attenuated tumor
growth as well as CAF number. In addition, maraviroc has
been found to be well tolerated by patients with acquired
immune deficiency syndrome without increasing theincidence of severe adverse effects [37]. Thus, several
groups have proposed the use of maraviroc for the treatmentof other diseases wherein CCR5 was presumed to be
implicated [19]. Given the crucial roles of the CCR5 axis
in CAF accumulation and function, maraviroc application
might therefore also be useful in the treatment of cancers
with an abundant number of CAFs.

pro-inflammatory tumor-promoting microenvironments[15], and by inducing neovascularization [16].
Furthermore, stromal markers are robustly associatedwith disease relapse in human CRC, which can arise froman increased frequency of tumor-initiating cells enhanced
by CAFs [17]. Thus, drug dampening CAFs might beable to supplement currently used anti-cancer treatments.
We previously unraveled the crucial role of the CCL3CCR5 axis in CAF accumulation in colitis-associatedcarcinogenesis and tumor formation arising from injectionof the mouse CRC cell line colon 26 either underneaththe skin or into the cecal wall [10]. Hence, in the current
study we investigated the therapeutic effects of a CCR5
antagonist, maraviroc, on preclinical models using amouse or a human CRC cell lines and demonstrated thatmaraviroc markedly attenuated tumor formation as well
as CAF accumulation with few effects on infiltrating
leukocyte numbers.
The identification of CCR5 as a crucial co-receptor
for human immunodeficiency virus (HIV) [18] promoted
the development of a CCR5 antagonist to be applied
to the treatment of HIV infection. Maraviroc, the only
CCR5 antagonist currently approved in various countries[19], binds to a site that is distinct from the proposed
recognition sites for chemokines and the HIV glycoprotein
gp120 [20]. The maraviroc binding site consists of 16
amino acids, 15 of which are common between mouse andhuman CCR5 proteins. Furthermore, several independentgroups have successfully used maraviroc to inhibit themouse CCR5 axis [21–27]. These observations incited us
to utilize maraviroc in these pre-clinical studies.
Initial immunofluorescence analysis detected
CCR5 expression in a subpopulation of T lymphocytes
associated with certain immune reactions, particularly
Th1 type reactions [28]. In addition, the interaction
between CCR5 and its ligand, CCL5, recruits Tregs [11]and tumor-associated macrophages [29, 30] as well as
increases the generation of MDSCs [12]. Thus, CCR5
blockade can counteract these immune suppressive cells
to enhance tumor immunity and to eventually reducetumor growth. However, CCR5 expression was barely
detected in infiltrating lymphocytes and macrophages
in our model and maraviroc did not markedly change
the macrophage, Treg, and MDSC numbers. Thus, it isunlikely that maraviroc acted by reducing the infiltrating
immunosuppressive cells to retard tumor growth in thismodel.
Several independent groups have observed that
CCR5 was expressed by some types of cancer cells
including breast [31], prostate [32], and gastric cancer[33]. Furthermore, in these studies, maraviroc directly
inhibited the in vivo invasiveness of these cancer cells andreduced subsequent tumor progression. In contrast, CCR5
blockade was reported to enhance the in vivo proliferationof breast cancer cells bearing wild-type p53 [34]. Thus,
the functionality of the CCR5 expressed by cancer
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MATERIALS AND METHODS
MiceSpecific pathogen-free female 7-week-old BALB/cmice (wild-type; WT mice) and male 7-week-old BALB/cnu mice (nude mice) were purchased from Charles River
Japan (Yokohama, Japan). All mice were maintained
under specific pathogen-free conditions. All the animal
experiments in this study were approved by the Committee
on Animal Experimentation of Kanazawa University andcomplied with the Guideline for the Care and Use ofLaboratory Animals of Kanazawa University.

Reagents and antibodies
Maraviroc was obtained from Sigma-Aldrich(St. Louis, MO, USA) (for in vitro experiments)
or GlaxoSmithKline (Brentford, UK) (for in vivo
experiments). Mouse CCL3 was obtained from Peprotech(Rocky Hill, NJ, USA). The following antibodies wereused as the primary antibodies for immunohistochemical
analyses: goat anti-CCR5 antibody (Santa Cruz
Biotechnology, Dallas, TX, USA), rat anti-Ly6G antibody(BD Biosciences, San Jose, CA, USA), rat anti-F4/80
antibody (Serotec, Kidlington, UK), mouse anti-α-SMA
antibody (Dako, Glostrup, Denmark), rabbit anti-type I
collagen antibody, rabbit anti-CD31 antibody and rabbit
anti-EGF antibody (Abcam, Cambridge, UK). The
following rat anti-mouse antibodies were used as theprimary antibodies for the flow cytometric analysis: anti48341

OncotargetCD11b antibody (BD Biosciences), anti-CD25 antibody(BioLegend, San Diego, CA, USA), anti-CD45 antibody(eBioscience, San Diego, CA, USA), anti-F4/80 antibody(eBioscience), anti-Foxp3 antibody (eBioscience),anti-Ly6G antibody (Gr-1) (Tonbo Biosciences, San
Diego, CA, USA), anti-MIP-1α antibody (R&D
Systems, Minneapolis, MN, USA), anti-CCR5 antibody(BioLegend), and isotype-matched control IgGs forindividual rat antibodies (BD Biosciences).

solution at a cell density of 1.25 × 106 and 2.5 × 107
cells/ml, respectively. The cell viability was confirmed
to be always >95% using a trypan blue exclusion
test. Prior to surgery, mice were anesthetized byan intraperitoneal injection of reagent containing
medetomidine (0.3 mg/kg), midazolam (4 mg/kg), andbutorphanol (5 mg/kg). A small incision was madealong the midline of the abdomen and the cecum wasexteriorized. Then, 40 µl cell suspensions of colon 26
and KM12C cells were injected into the cecal wall of
WT and nude mice, respectively, using a 30-gauge
needle. After surgery, the abdominal muscle wall was
closed using surgical silk sutures. Mice also received
an intraperitoneal injection of the anti-medetomidine
inhibitor, atipamezole (0.3 mg/kg). From 2 days
after the tumor injection, mice were administeredmaraviroc at a dose of 30 mg/kg every 2 days. At
the indicated time intervals from the inoculation,
cecum was removed for the determination of tumor
volume, immunohistochemical analysis, and total RNA
extraction followed by qRT-PCR. Tumor volumes were
calculated according to the following equation: π × a ×b × c/6, where a, b, and c indicate the width, depth, andheight of the tumor, respectively.

Cell lines
A murine CRC cell line, colon 26 (CRL-2638;
American Type Culture Collection (ATCC), Manassas, VA,USA) and a human CRC cell line, KM12C (a gift from Dr.
I. J. Fidler, M.D. Anderson Cancer Center) were maintainedin RPMI 1640 medium supplemented with 10% fetal
bovine serum (FBS). A mouse fibroblast cell line, NIH3T3(CRL-6361; ATCC) was maintained in Dulbecco’s modified
Eagle’s medium supplemented with 10% FBS. These cells
were cultured at 37°C under 5% CO2.

In vitro cell proliferation of CRC cell lines
Cell suspensions (2 × 103 cells /100 μl) were added
to each well of 96-multi-well plates and incubated at 37°C
for 24 h. The cells were then treated with the indicatedconcentrations of maraviroc for 72 h. The cell viability
was determined using the cell counting kit-8 (Dojindo
Co. Ltd., Kumamoto, Japan), which is based on thedetermination of the extracellular WST-8 reduction by
NADH produced in the mitochondria resulting in soluble
formazan. The ratios of cell proliferation were determinedby comparison to the number of untreated cells.

Histological and immunohistochemical analysis
Resected tumor tissues were fixed in 10%
formaldehyde and embedded in paraffin. The sections
were cut at 4-μm thickness and stained with hematoxylin
and eosin solution. In parallel, 5-μm-thick sections weregenerated for immunohistochemical analysis. For antigen
retrieval of paraffin sections, the deparaffinized slides
were either autoclaved in 10 µM citrate buffer (pH 6.0)
for 15 min at 121°C (for staining with anti-type I collagen,anti-CD31 or anti-α-SMA), treated with 0.1% proteinase
K solution for 15 min at 37°C (for staining with antiEGF or anti-CCR5), or treated with 0.1% trypsin solution
for 15 min at 37°C (for staining with anti-F4/80 or antiLy6G). Endogenous peroxidase activity was blocked
using 0.3% H2O2 for 15 min, followed by incubation with
Blocking One Histo (Dako Japan, Kyoto) for 15 min. The
sections were further incubated with the optimal dilutionof the antibodies overnight in a humidified box at 4°C.
The resultant immune complexes were detected by the
ABC Elite kit (Vector Laboratories, Burlingame, CA,
USA) and peroxidase substrate 3-3’-diaminobenzidinekit (Vector), according to the manufacturer’s instructions.
Positive signals or areas were measured on 5 randomly
chosen visual fields at ×100 magnification by means of
Image J (National Institutes of Health, Bethesda, MD,
USA). For double-color immunofluorescence analysis,
Alexa Fluor 594 donkey anti-mouse, Alexa Fluor 488
donkey anti-goat antibody, or Alexa Fluor 488 donkey
anti-rabbit antibody (Invitrogen, Carlsbad, CA, USA)
were used as secondary antibodies. Immunofluorescence

In vitro experiments using NIH3T3 cells
Sub-confluent NIH3T3 cells were seeded at a celldensity of 3 × 105 cells/ml in each well of a 24-multi-wellplate. After 24 h later, the cells were treated with 100 nM
maraviroc for 1 h. Thereafter, mouse CCL3 was added toeach well at 100 ng/ml. The cells were harvested to extract
total RNA at 6 h after the addition of CCL3 as described
below. The resultant total RNA was subjected to qRTPCR. For in vitro cell migration analysis, the indicatedconcentrations of mouse CCL3 and 50,000 cells wereplaced in the lower and upper chambers of 24-well transwell chambers (8 μM pore size; Costar, Cambridge, MA,
USA), respectively, in the presence or absence of 100 nM
maraviroc, and were incubated at 37°C for 24 h in 5% CO2.
After gently removing the upper chambers, the numbers ofcells that had passed to the lower chambers were counted.

Orthotopic tumor formation
Subconfluent colon 26 and KM12C cells werecollected and resuspended in Hank’s balanced salt
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For intracellular MIP-1α/CCL3 staining, isolated single
cell suspensions were incubated in serum-free RPMI
1640 medium supplemented with 0.1% GolgiStop reagent(BD) for 4 h. Subsequently, intracellular CCL3 wasstained with PE-conjugated anti-CCL3 antibody assisted
by an Intracellular Cytokine Staining Starter kit (BD).
Intracellular staining for Foxp3 was performed using a
Foxp3 Staining kit (BD). Expression of each molecule wasdetermined using FACSCanto II (BD) and analyzed with
FlowJo software (Tree Star, Ashland, OR, USA).

qRT-PCR analysis
Total RNAs were extracted from the cell lines ortumors using the RNeasy Mini Kit (Qiagen, Venlo, The
Netherlands). After treatment with RNase-free DNase I(Qiagen), 2 μg total RNA was reverse-transcribed using theSuperScript III First-Strand Synthesis System (Invitrogen)to obtain cDNA. qPCR was performed on the obtained
cDNA using the StepOne real-time PCR system (Applied
Biosystems, Foster City, CA, USA) using the Quantifast
SYBR Green PCR Master mix (Applied Biosystems) andthe primers listed in Table 1. Expression levels of the target
genes were analyzed using the ΔΔCT comparative thresholdmethod. The glyceraldehyde-3-phosphate dehydrogenase(Gapdh) gene was used as an internal control.

Enzyme-linked immunosorbent assay (ELISA)
The tissues collected at the indicated timeintervals were homogenized with a lysis buffer (20mM Tris-HCl, pH 7.6; 150 mM NaCl; 1% Triton;and 1 mM EDTA) containing Complete Protease
Inhibitor Cocktail (Roche-Diagnostics, Roswell,
GA, USA). Homogenates were centrifuged toobtain protein lysates. After the determination ofprotein concentration using the BCA Protein Assaykit (Pierce, Rockford, IL, USA), CCL3 and CCL4
levels were measured in the protein lysates using thespecific ELISA kits (R&D Systems), according to the
manufacturer’s instructions.

Flow cytometry
Tumor tissues were obtained from the cecum. Afterremoving the epithelial cells, the tissues were further
cut into small pieces and incubated with RPMI 1640
containing 1 mg/ml collagenase type I and 40 µg/ml
DNase I for 20 min at 37°C with shaking for 180 rpm.
The resulting digestion mixtures were filtered through a
100-µm cell strainer (Corning, Cambridge, MA, USA)to achieve single cell suspensions. Isolated single cellsuspensions were stained with various combinations offluorescent dye-conjugated antibodies in MACS buffer.

Statistical analysis
The data were statistically analyzed using themethods indicated in each figure legend. p values less than
0.05 were considered statistically significant.

Table 1: Nucleotide sequence of the primers used for qRT-PCR
Gene nameSpecies

Size

Gapdh

mouse

55

catggccttccgtgttccta

gcggcacgtcagatcca

Ccr5

mouse

61

catccgttccccctacaaga

ggaactgacccttgaaaatcca

Ccl3

mouse

57

gctgacaagctcaccctctgt

ggcagtggtggagaccttca

Ccl4

mouse

57

cagcaccaatgggctctga

gccgggaggtgtaagagaaac

Ccl5

mouse

80

tccaatcttgcagtcgtgtttg

tctgggttggcacacacttg

Fgfb

mouse

127

gacccacacgtcaaactacaactc

ctgtaacacacttagaagccagcag

Ctgf

mouse

232

ggtgagtccttccaaagcag

ggccaaatgtgtcttccagt

Egf

mouse

111

tgcctcagaaggagtgggtta

gtgttccaagcgttcctgaga

Egfr

mouse

204

gaactgggcttagggaactgc

cattgggacagcttggatcac

Ereg

mouse

166

taccgccttagttcagatgg

acatcgcagaccagtgtagc

Hb-egfmouse

111

gcaaatgcctccctggttac

ctacagccaccacagccaaga

Hgf

mouse

57

tcggataggagccacaagga

ccgaggccagctgcaat

Pdgf

mouse

154

gcaccaacgccaacttcct

atgggcttctttcgcacaat

Vegf

mouse

234

ctactgccgtccgattgaga

catctgctgtgctgtaggaag

www.impactjournals.com/oncotarget

Forward primer (5’-3’)

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Reverse primer (5’-3’)

Oncotarget

CONFLICTS OF INTEREST

12. Zhang Y, Lv D, Kim HJ, Kurt RA, Bu W, Li Y and Ma
X. A novel role of hematopoietic CCL5 in promoting
triple-negative mammary tumor progression by regulating
generation of myeloid-derived suppressor cells. Cell Res.
2013; 23:394-408.

No potential conflicts of interest were disclosed.

GRANT SUPPORT

13. Dube PE, Yan F, Punit S, Girish N, McElroy SJ, Washington
MK and Polk DB. Epidermal growth factor receptor inhibits
colitis-associated cancer in mice. J Clin Invest. 2012;
122:2780-2792.

This work was supported by a Grant-in-Aid forYoung Scientists (B) 15K18406 from the Japan Society
for the Promotion of Science (JSPS).

14. Flaberg E, Markasz L, Petranyi G, Stuber G, Dicso F,
Alchihabi N, Olah E, Csizy I, Jozsa T, Andren O, Johansson
JE, Andersson SO, Klein G and Szekely L. High-throughput
live-cell imaging reveals differential inhibition of tumor
cell proliferation by human fibroblasts. Int J Cancer. 2011;
128:2793-2802.

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