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European Journal of Cancer (2013) xxx, xxx xxx

Available at www.sciencedirect.com

journal homepage: www.ejcancer.com

Crosstalk between colon cancer cells and macrophages


via inammatory mediators and CD47 promotes tumour cell
migration
Yuan Zhang, Wondossen Sime, Maria Juhas, Anita Sjolander
Division of Cell and Experimental Pathology, Department of Laboratory Medicine, Clinical Research Centre, Lund University,
Skane University Hospital, SE-205 02 Malmo, Sweden

KEYWORDS
Colon cancer
Tumour-associated
macrophage
CD47
Cell migration

Abstract Tumour-associated macrophages (TAMs) of the M2 phenotype are present in the


stroma of many tumours and are frequently associated with the progression of several types
of cancer. We investigated the role of M2 macrophages in colon cancer progression and found
that human colon cancer tissue had elevated numbers of CD68+ (macrophage marker) cells
and CD206+ (M2 macrophage marker) cells and increased CD47 expression. To explore
potential interplay between colon cancer cells and M2 macrophages, we differentiated the
monocyte cell line THP-1 into M1 and M2 macrophages (CD206high and Th2 cytokine-secreting cells), respectively. M2 macrophages migrated faster than M1 macrophages towards
SW480-conditioned medium. Similarly, M2 macrophage-conditioned medium induced
SW480 cell migration and CD47 expression. Factors released by macrophages were involved
in this induction. In addition, SW480 cells migrated faster when co-cultured with M2 macrophages. Inhibition of CD47 with blocking antibodies or siRNA signicantly reduced the
migration of SW480 cells in the presence of M2 macrophages. This effect was further
decreased via blocking antibodies against the CD47 ligand signal-regulatory protein a
(SIRPa). Additionally, cancer cells also secreted signicant levels of IL-10, thereby promoting
M2 macrophage differentiation. These ndings indicate that a TAM-enriched tumour microenvironment promotes colon cancer cell migration and metastasis.
2013 Elsevier Ltd. All rights reserved.

Corresponding author: Address: Division of Cell and Experimental Pathology, Department of Laboratory Medicine, CRC, Lund University,
Skane University Hospital, Jan Waldenstrom gata 35, Building 91, Floor 11, SE-205 02 Malmo, Sweden. Tel.: +46 40 391168; fax: +46 40 391177.
E-mail address: anita.sjolander@med.lu.se (A. Sjolander).

0959-8049/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ejca.2013.06.005

Please cite this article in press as: Zhang Y. et al., Crosstalk between colon cancer cells and macrophages via inammatory mediators and CD47
promotes tumour cell migration, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.005

Y. Zhang et al. / European Journal of Cancer xxx (2013) xxxxxx

1. Introduction
Colorectal cancer is the third most commonly diagnosed cancer in the world. Patients suering from inammatory bowel diseases, such as Crohns disease and
ulcerative colitis, have an elevated risk of developing
colon cancer.1 In recent years, the tumour microenvironment has been highlighted as an important hallmark of
cancer.2,3 In tumour microenvironments, macrophages
represent a major inammatory component of the stroma
and aect many aspects of the neoplastic tissue.4 Tumourassociated macrophages (TAMs) play an important role
in cancer progression, and accordingly, high levels of
macrophage inltration of the tumour tissues are associated with poor prognosis in cancer patients.4 Intratumoural macrophages that express the macrophage markers
CD68 and CD163 are enriched in colon carcinoma and
may promote tumourigenesis5; however, the mechanism
of such promotion remains unclear. Evidence indicates
that intratumoural macrophages are recruited by tumour
cells.6 The tumour microenvironment is thought to
induce the tumour-promoting M2 macrophage phenotype.7 Therefore, specic visualisation of M2 macrophages in colon cancer tissues is crucial.8
Macrophages produce a wide array of cytokines and
lipid mediators, such as prostaglandins and leukotrienes
(LTs).9 M1 macrophages are pro-inammatory and
potentiate Th1 responses by secreting high levels of
interleukin (IL)-1, IL-12, interferon (IFN)-c and other
Th1 cytokines, whereas M2 macrophages secrete high
levels of IL-8 and Th2 cytokines, such as IL-4, IL-10
and IL-13, which contribute to the maintenance of an
immunosuppressive microenvironment.10 It has been
documented that the prostaglandin E2 increases migration and invasion of colon cancer cells.11 The proinammatory cysteinyl leukotriene D4 (LTD4) has also
been implicated in colon cancer progression,12 and we
have shown previously that LTD4 increases proliferation, migration and survival in colon cancer cells via
CysLT1 receptor (CysLT1R) signals.1315 Equally
important, we have shown that colon cancer patients
with high CysLT1R expression in their tumour tissues
have a poor prognosis.16 Therefore, we hypothesise that
LTD4 released from M2 macrophages inuences colon
cancer cell behaviour.
Apart from soluble factors, cell surface-bound molecules regulate the interaction between intratumoural
macrophages and colon cancer cells. One such molecule
that was recently shown to be commonly expressed on
cancer cells is CD47.17 CD47 was rst described as an
anti-phagocytosis molecule based on its interaction with
signal regulatory protein alpha (SIRPa), which is
expressed on the surface of macrophages.18 In the context of cancer, such a mechanism could enable cancer
cells to escape the immune system.17 The interaction
between CD47 and SIRPa has also been suggested to

contribute to cell migration. For example, CD47 has


been shown to control the migration of canine kidney
epithelial cells19 and intestinal epithelial cells grown on
a collagen I-coated surface.20 Inhibition of CD47
SIRPa interactions with a CD47 blocking antibody
may be a potential therapeutic approach17; however,
the precise role of CD47 in colon cancer cell migration
and metastasis remains to be determined.
In this study, we report that M2-like macrophages
secrete immunosuppressive cytokines to create a microenvironment that promotes colon cancer cell growth and
migration. In addition, colon cancer cells exhibit elevated CD47 levels, which can be inuenced by macrophages, and secrete high levels of IL-10, enhancing the
M2 macrophage phenotype. Increased CD47SIRPa
signalling between colon cancer cells and M2-like macrophages promotes cancer cell migration. These ndings
highlight the importance of TAM activity in the colon
cancer microenvironment.
2. Materials and methods
2.1. Cell culture, stimulation and dierentiation
The human colon cancer cell line SW480,21 HCT-116
and human monocyte cell line THP-1 (obtained from
ATCC) were cultured following the suppliers instructions. SW480 cells were stimulated with 20 ng/ml IL-4,
80 nM LTD4, 100 ng/ml IL-8 or M2 macrophage-conditioned medium for 24 h at 37 C after overnight serum
starvation. THP-1 cells were dierentiated by treatment
with 100 nM phorbol-12-myristate-13-acetate for 7
days. The adhered cells were further dierentiated with
100 ng/ml lipopolysaccharide for 72 h and 20 ng/ml
IFN-c during the last 48 h (M1) or 20 ng/ml IL-4 for
48 h (M2; Fig. 2A). For multiplex assays, enzyme-linked
immunosorbent assays (ELISAs), and conditioned medium collecting, the dierentiated macrophages were
washed with serum-free medium to remove the dierentiation factors and cultured in 1.5% foetal bovine serum
(FBS)-containing medium for an additional 3 days.
2.2. Patient samples
Formalin-xed and paran-embedded colon cancer
and control colon specimens from colorectal cancer
patients were obtained from the archives of the Department of Pathology at Malmo University Hospital.16,21
Tissues from 72 patients with varying grades and stages
of disease were included. Staging of the tumours was
done using Dukes classication.22 Fresh biopsies were
obtained for mRNA analysis. The biopsy samples were
placed in RNAlater (Qiagen, Hilden, Germany) and frozen by submersion in liquid nitrogen. The matched control samples from normal colon tissues were surgical
specimens from the same patients. Specimens were

Please cite this article in press as: Zhang Y. et al., Crosstalk between colon cancer cells and macrophages via inammatory mediators and CD47
promotes tumour cell migration, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.005

Y. Zhang et al. / European Journal of Cancer xxx (2013) xxxxxx

Fig. 1. CD68 and CD206 expression in colon cancer tissues. (A) mRNA expression of CD68 and CD206 in normal and tumour tissues from colon
cancer patients. (B and E) CD68 and CD206 immunohistochemistry staining. (C, D, F, and G) Number of CD68+ and CD206+ cells in tissues from
colon cancer patients. (H) KaplanMeier survival curve using log-rank test. Numbers of CD206+ cells in tumour tissues were compared to normal
tissues. The results of mRNA expression analysis are shown as the mean standard error of the mean (SEM) of four separate experiments.
*
P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Students t-test).

Please cite this article in press as: Zhang Y. et al., Crosstalk between colon cancer cells and macrophages via inammatory mediators and CD47
promotes tumour cell migration, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.005

Y. Zhang et al. / European Journal of Cancer xxx (2013) xxxxxx

Fig. 2. Dierentiation and cytokine proles of M1 and M2 macrophages derived from the human monocyte cell line THP-1. (A) Schematic
diagram showing the in vitro dierentiation of THP-1 cells into M1 and M2 macrophages. Images show CD68 expression during dierentiation. (B)
The mRNA expression levels of CD68 and CD206. (C) Histogram overlay from ow cytometry showing cell surface expression of CD68 and
CD206 in M1- and M2-like macrophages. (D) Signal-regulatory protein a (SIRPa) expression in M1- and M2-like macrophages. (E) Cytokine
proles of SW480 cells, M1 or M2 macrophages as well as of co-cultures of SW480 with M1 or M2 macrophages by multiplex assay. The results are
shown as the mean standard error of the mean (SEM) of 39 separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (column statistics for B
and D, two-tailed Students t-test for E).

Please cite this article in press as: Zhang Y. et al., Crosstalk between colon cancer cells and macrophages via inammatory mediators and CD47
promotes tumour cell migration, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.005

Y. Zhang et al. / European Journal of Cancer xxx (2013) xxxxxx

obtained with informed consent after ethical approval


was granted by the Ethics Committee of Lund
University.
2.3. Tumour xenograft study
The Regional Ethics Committee for Animal Research
at Lund University, Sweden (M205-10) approved the
present animal study. Female 6- to 8-week-old athymic
nude mice (BalbCnu/nu) were purchased from Taconic
Europe A/S (Ry, Denmark). To generate subcutaneous
human colon cancer xenografts, 2.5  106 low-passage
HCT-116 cells suspended in 100 ll phosphate-buered
saline (PBS) were injected into both anks of the mice
(21 mice in total). Twenty-one days after tumour cell
inoculation all mice were sacriced, and the tumours
were removed. Tumour tissues were immediately xed
in 10% buered formalin and then embedded in paran
for further immunohistochemical analysis.
2.4. Immunohistochemistry
Formalin-xed, paran-embedded archival colon
cancer human specimens were stained with CD68
(1:50; Biolegend, San Diego, CA, United States of
America (USA)), CD206 (1:50; BD Biosciences, San
Jose, CA, USA), CD47 (40 lg/ml; a kind gift from Dr.
P.A. Oldenborg), SIRPa (1:500; LifeSpan BioScience,
Inc., Seattle, WA, USA) antibodies. The mice xenographs were stained with the F4/80 (1:100; AbD Serotec,
Raleigh, NC, USA) antibody. All stained tissues were
visualised by incubation with secondary peroxidase-conjugated antibodies. After immunostaining, all slides
were manually counterstained with Mayers haematoxylin. Slides were scanned with the ScanScope CS (Aperio,
Vista, CA, USA) at 10 and 40 magnication. Numbers of CD68+ and CD206+ cells were counted with
NIS-Elements Advanced Research software (Nikon,
Tokyo, Japan) and statistically analysed with SPSS software. The total F4/80 positive stained area per tumour
was measured with Aeprio ImageScope software (Aperio, Vista, CA, USA).

2.6. Immunouorescent staining


THP-1 cells were attached to coverslips by centrifugation at 1000 rpm for 5 min. Fixation and blocking were
performed as previously described.20 The cells were
incubated with SIRPa, CD68, CD206 or mouse immunoglobulin G (IgG) antibodies (1:100) for 1 h, washed
with PBS and incubated with Alexa 488 or Alexa 546
goat anti-mouse secondary antibody (1:500) for 1 h.
For double staining, an extra blocking step was performed. After washing with PBS, the cells were incubated with DAPI (1:1000) for 3 min and mounted in
uorescent mounting medium (Dako, Glostrup, Denmark). The slides were photographed with a Nikon
Eclipse 80i microscope using a PlanApo 60 objective
and NIS-Elements Advanced Research software
(Fig. 2B), an Olympus FluoView FV10i Confocal
Microscope using a 60 objective (Olympus Corporation, Tokyo, Japan; Fig. 3B), or a Zeiss LSM 700 confocal microscope (Carl Zeiss Microscopy GmbH, Jena,
Germany; Fig. 6A).
2.7. Flow cytometry
The dierentiated M1 and M2 macrophages were
detached using 0.02% versene and were washed twice
in 0.5% bovine serum albumin (BSA)/PBS before blocking the human FcRs with 20 lg/ml heat-aggregated
human IgG. Next, 0.5  105 cells were suspended in
100 ll 0.5% BSA/PBS and incubated with 2 lg/ml
CD68 antibody followed by incubation with Alexa 488
goat anti-mouse secondary antibody (30 min each at
4 C). Cells were incubated with 20 ll allophycocyanin-conjugated CD206 (BD Biosciences) or 5 ll SIRPa
antibody (eBioscience). Cells were examined with a
FACS-Calibur using the software Cell Quest (Becton
Dickinson, San Jose, CA, USA) and analysed using
FCS Express Version 4 (De Novo Software, Los Angeles, CA, USA). Forward and side scatter gates were set
to include all viable cells.
2.8. Co-culture of M1 or M2 macrophages with SW480
cells

2.5. RT-PCR
RNA from cells and tissue samples was isolated
following the protocol of the Qiagen RNeasy Plus
Mini Kit.21 The following primers were obtained
from Applied Biosystems (Cambridge, United
Kingdom (UK)): CD68 (Hs00154355_m1), CD206
(Hs00267207_m1), CD47 (Hs00964717_m1), and SIRPa
(Hs00388955_m1). Amplication was performed in a
Mx3005P system (Agilent Technologies, Inc., CA,
USA), and reactions were analysed with MxPro software and normalised against the housekeeping gene
HPRT1.

SW480 cells were counted before adding pre-dierentiated M1 or M2 macrophages at a ratio of 1:1 or 2:1
and cultured in 1.5% FBS-containing medium for 72 h.
The conditioned medium was collected for a multiplex
assay or ELISA. Medium containing 1.5% FBS was
used as a negative control.
2.9. Measurement of secreted cytokines by multiplex
assay
To remove cell debris, the conditioned medium
was collected and centrifuged at 1000 rpm for 5 min.

Please cite this article in press as: Zhang Y. et al., Crosstalk between colon cancer cells and macrophages via inammatory mediators and CD47
promotes tumour cell migration, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.005

Y. Zhang et al. / European Journal of Cancer xxx (2013) xxxxxx

Fig. 3. Education of M1-like macrophages. (A) Histogram overlay showing cell surface expression of signal-regulatory protein a (SIRPa) in M1
macrophages, M2 macrophages and M1 macrophages stimulated with IL-10 for 72 h and analysed by ow cytometry. (B) Representative
immunouorescence images showing expression and localisation of CD206 (green) and SIRPa (red) in M2 and M1 macrophages with/without
IL-10 stimulation. (C) M1 macrophages co-cultured with SW480 cells. DAPI is shown in blue. Scale bar is 20 lm. (For interpretation of the
references to colour in this gure legend, the reader is referred to the web version of this article.)

Electrochemiluminescence assays were performed on


macrophage-conditioned medium in duplicate using a
10-plex human TH1/TH2 detection kit (Meso Scale
Discovery, Gaithersburg, MD, USA) according to the

manufacturers instructions. The plates were read using


the Meso Scale Discovery SECTOR Imager 6000 and
analysed using Discovery Workbench and SoftMax
PRO 4.0 software.

Please cite this article in press as: Zhang Y. et al., Crosstalk between colon cancer cells and macrophages via inammatory mediators and CD47
promotes tumour cell migration, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.005

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2.10. Transfection with CD47 siRNA oligomers


SW480 cells were cultured for 3 days to 5060% conuence. The growth medium was aspirated, and the cells
were washed with OPTI-MEM I (Invitrogen Corp.,
Carlsbad, CA, USA). Next, 5 ml of OPTI medium containing 75 nM siRNA against CD47 (ID #: 2811,
145978 and 145979) or a scrambled control siRNA
(Ambion, Cambridgeshire, UK) was added to the cells
along with lipofectamine 2000. After 4 h, the transfection medium was diluted with RPMI 1640 medium
(10% FBS) without antibiotics, and the cells were
allowed to grow for an additional 72 h with a medium
change after 24 h.
2.11. Western blotting
Whole cell lysates were prepared, and Western blotting was performed as described.13 The polyvinylidene
diuoride (PVDF) membranes were blocked for 1 h at
room temperature with either 4% BSA/PBS-T (0.05%
Tween 20) or 5% non-fat dried milk. The membranes
were incubated overnight at 4 C with anti-CD47
(MEM122, 1:1000; EXBIO, Prague, Czech Republic),
stripped and reprobed with anti-b-actin (1:10,000) antibodies. The membranes were washed and incubated for
1 h at room temperature with HRP-conjugated secondary antibodies diluted at 1:10,000. Proteins were
detected after incubation with an Immun-Star Western
Chemiluminescence Kit (BioRad, Hercules, CA, USA)
using a Bio-Rad ChemiDoc XRS+ System. Bio-Rad
Image Lab software was used for densitometric analysis,
and the value obtained from the non-stimulated control
was set to 100.
2.12. Wound healing assay
SW480 cells were serum starved for 2 h and incubated
with or without 2 lg/ml IL-8 receptor CXCR1 antibody
(Abcam, Cambridge, UK), 50 lM CysLT1R inhibitor
ZM198,615 (AstraZeneca, R&D Lund, Sweden), or
20 lg/ml anti-CD47 antibody B6H1220 for 30 min,
15 min or 20 min, respectively. The wound healing assay
was performed as described previously.20
For the co-cultured cell wound healing assay, M2
macrophages were dierentiated in the wells of an ibidi
culture-insert (ibidi, Martinsried, Germany). On day 4,
2.5  106 SW480 cells were added to the dishes outside
of the insert. Thereafter, SW480 cells and macrophages
were cultured for an additional 5 days. On day 9, the
culture medium from the macrophages was aspirated
before the insert was carefully removed. In some experiments, transfected SW480 cells were used and M2 macrophages in the ibidi wells were treated with 10 lg/ml
anti-SIRPa antibody (Biolegend, San Diego, CA,
USA) for 30 min. SW480-conditioned medium was col-

lected, centrifuged to remove cell debris and added back


to the dishes. Non-adherent cells were gently washed in
PBS. The cells were allowed to migrate for 24 h at 37 C.
Pictures were taken20 with a Nikon DS-Fi1 microscope
using a 10 objective and analysed with NIS-Elements
Basic Research software. The area of the wound was
measured with Adobe Photoshop CS4 software.
2.13. Boyden chamber assay
M1 and M2 macrophages were detached by the addition of 0.02% versene and collected. Approximately
5  104 cells were added to the upper well of a Boyden
chamber (Neuro Probe, Gaithersburg, MD, USA).
The lower well contained supplemented RPMI 1640
(10% FBS) or SW480-conditioned medium. The two
wells were separated by an 8.0-lm polycarbonate PVDF
membrane. After a 4-h incubation at 37 C, cells that
had not migrated were removed, and the membrane
with migrated cells was xed and stained as described
previously.20 The membrane was washed, the remaining
dye solubilised in 10% sodium dodecyl sulphate and the
absorbance measured at 590 nm.
2.14. Statistical analyses
SPSS software 16.0 was used for all immunostaining
analyses. Univariate survival analysis was performed by
KaplanMeier analysis with a log-rank test to determine
the risk of death. Survival time was measured from the
date of surgery to the date of death or 80 months of follow-up. The death information is from the Swedish
Cause of Death Registry and the Swedish Cancer Register. The overall survival was calculated as colon cancerspecic death. Deaths due to other causes were censored
at the time of death. Prizm software 5.0d (GraphPad
Software, San Diego, CA, USA) was used for other statistical analyses. All the data are presented as the
mean standard error of the mean (SEM), and statistical signicance was determined as P < 0.05 by a twoway analysis of variance (ANOVA; labelled #), Column
statistics or a two-tailed Students t-test (labelled ). All
means were calculated from data from at least three
independent experiments.
3. Results
3.1. Macrophage content in human colon cancer tissue
Increased macrophage inltration has been detected
in many dierent solid tumours.23 To quantify and distinguish the phenotype of these macrophages, expression of CD68, which is expressed at high levels on
macrophages, and CD206, which is highly expressed
on M2 macrophages, was examined by Q-PCR and
compared between normal and tumour tissues from

Please cite this article in press as: Zhang Y. et al., Crosstalk between colon cancer cells and macrophages via inammatory mediators and CD47
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colon cancer patients. Expression of CD206 was 1.8-fold


higher in tumour tissues than in matched normal tissues,
while CD68 expression was similar in both tissue types
(Fig. 1A).
To examine macrophage inltration in colon cancer,
tissues from patients were stained for CD68. In normal
tissues, the CD68+ cells were primarily located outside
the epithelial cell layer, whereas in the tumour tissue,
these cells were observed mostly between the epithelial
cancer cells (Fig. 1B). The number of CD68+ cells was
determined using a colon cancer tissue array, which contains 72 pairs of normal mucosa and tumour tissue
patient samples. In this experiment, tissues from 62 of
the 72 patients were grouped, according to Dukes stage
classication. Signicantly more macrophages were
observed in tumour tissues compared with normal tissues (Fig. 1C), especially in the late stage of colon cancer
classied as Dukes C (Fig. 1D).
Similarly, staining of the same colon cancer tissue
array with CD206 antibody revealed signicantly
increased numbers of CD206+ cells in the tumour tissues
as compared with normal tissues (Fig. 1F and G), and
these cells were located between the epithelial cancer
cells (Fig. 1E). This nding indicated that the tumour
area contained more M2 macrophages than the normal
area. Additionally, we observed that a higher number of
CD206+ cells in the tumour than in the normal tissue
tends to confer a poorer prognosis (Fig. 1H).
To assess the above results from both the in vitro
experiments and human colon cancer tissue we next
set up a mouse xenograft-model with human colon cancer cells. We found, in good agreement with our human
in vivo results, a higher content of macrophages (F4/80
positive cells) in the larger tumours as compared
with the smaller tumours (Appendix Supplementary
Fig. S1). These results further stress the importance of
TAMs in colon cancer progression.
3.2. M1- and M2-like macrophages dierentiated from
the human monocyte cell line THP-1
To establish an in vitro model, the human monocyte
cell line THP-1 was dierentiated into M2 or M1 macrophages (Fig. 2A). THP-1 cells that normally grow in suspension and have a rounded morphology became
attached and spread more during dierentiation.
Expression of CD68 increased during dierentiation,
indicating that the cells became more macrophage-like
(Fig. 2A). To conrm the phenotype of these macrophages, the mRNA levels of macrophage markers were
investigated. Both M1 and M2 macrophages expressed
similar levels of CD68 mRNA, but the mRNA levels
of CD206 were signicantly higher (23-fold) in the M2
population (Fig. 2B). A similar expression pattern for
macrophage marker expression was observed at the protein level using ow cytometry (Fig. 2C).

In addition, signicantly higher expression of SIRPa


was observed at both the mRNA (2.5-fold) and protein levels (3.8-fold) in the dierentiated M2 macrophages compared to the dierentiated M1 macrophages (Figs. 2D
and 3A). This result suggests that colon cancer cells that
express high levels of CD47 interact with M2 macrophages
via an interaction between CD47 and SIRPa.
3.3. Cytokine proles of THP-1-derived macrophages
Macrophages derived from various tissue sites are
known to exhibit dierent cytokine proles.10 Thus, we
measured the levels of secreted cytokines in conditioned
media from cells grown under dierent conditions as
described in Section 2. The levels of Th2 cytokines,
including IL-4 (122 pg/ml), IL-10 (118 pg/ml) and IL-8
(5531 pg/ml), were signicantly higher in the M2 macrophage-conditioned medium compared to the M1 macrophage-conditioned media (Fig. 2E). IL-4 secretion was
observed in M2 but not M1 macrophage-conditioned
media. IL-8 expression was dramatically increased in
the conditioned media from M2 macrophages co-cultured with SW480 cells than in media from M2 macrophages alone (from 673 to 4886 pg/ml). Moreover, the
Th1 cytokines, including IL-1b, IFN-c and tumour
necrosis factor (TNF)-a, were more highly expressed
in the M1 macrophage conditioned medium. Interestingly, the IL-10 concentration in the SW480-conditioned
medium (204 pg/ml) was much higher than that in the
macrophage-conditioned medium.
The cytokine proles from the M1 and M2 macrophages conrmed the macrophage dierentiation eciency, and these data highlighted several cytokines
known to inuence the behaviour of the tumour microenvironment. IL-10 is an immunosuppressive cytokine
that is known to be important in the dierentiation of
M2 macrophages.24 Therefore, we investigated the role
of IL-10 in macrophage polarisation.
3.4. IL-10 secreted from SW480 cells polarise M1
macrophages
SW480 cancer cells secreted signicant amounts of
IL-10 (Fig. 2E). We therefore hypothesised that M1
macrophages dierentiate into M2 macrophages in the
cancer cell milieu. We stimulated dierentiated M1 macrophages with IL-10 for 72 h and followed changes in
expression of CD206 and SIRPa. Fluorescent staining
of CD206 was increased at the cell membrane of stimulated M1 macrophages, similar to that observed in differentiated M2 macrophages (Fig. 3B). Expression of
SIRPa increased from a mean uorescent intensity of
178 to 249 in M1 macrophages stimulated with IL-10
for 72 h (Fig. 3A). This increase in SIRPa expression
was conrmed by immunouorescence staining in
IL-10-stimulated cells (Fig. 3B). Furthermore,

Please cite this article in press as: Zhang Y. et al., Crosstalk between colon cancer cells and macrophages via inammatory mediators and CD47
promotes tumour cell migration, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.005

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immunouorescent staining indicated that CD206 and


SIRPa expression were increased in M1 macrophages
co-cultured with IL-10-releasing SW480 cancer cells
for 72 h (Fig. 3C). These ndings support the hypothesis
that M1 macrophages are prone to become M2-like
when exposed to factors, such as IL-10, that are released
by SW480 cells.

3.5. Migration of SW480 colon cancer cells is induced by


M2 macrophage-derived factors
Tumour cells are known to recruit macrophages6;
thus, we performed a Boyden chamber assay to investigate the ability of SW480 colon cancer cells to attract
macrophages. Signicantly more M2 macrophages than

Fig. 4. Migration of SW480 colon cancer cells stimulated by M2 macrophage-derived factors. (A) Boyden chamber assay demonstrates that M1and M2-like macrophages migrate towards SW480-conditioned medium. (B) Wound healing assay involving SW480 cells with or without M2
macrophage-conditioned medium at indicated time points. (C) Wound healing assay with SW480 cells pre-treated with IL-8 receptor CXCR1
blocking antibody, CysLT1R inhibitor ZM198,615, or CD47 blocking antibody B6H12 and stimulated with M2 macrophage-conditioned medium.
(D) CD47 expression in SW480 cells upon IL-4, IL-8, LTD4 or M2 macrophage-conditioned medium stimulation after 6 or 24 h. The results are
shown as the mean standard error of the mean (SEM) of 37 separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (column statistics for D,
two-tailed Students t-test for A and B), #P < 0.05 (two-way ANOVA).

Please cite this article in press as: Zhang Y. et al., Crosstalk between colon cancer cells and macrophages via inammatory mediators and CD47
promotes tumour cell migration, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.005

10

Y. Zhang et al. / European Journal of Cancer xxx (2013) xxxxxx

M1 macrophages migrated towards the SW480 cancer


cell-conditioned medium (Fig. 4A). The tumour microenvironment is rich with various soluble and non-soluble factors, which are believed to be essential for
tumour progression. In this study, M2 macrophages
were shown to release cytokines, and inammatory
mediators, and thus, we examined the contribution of
these dierent factors to the interaction between M2
macrophages and colon cancer cells as well as to cell
migration. In a wound healing assay, SW480 cells were
stimulated with M2 macrophage-conditioned medium,
and wound closure was measured at dierent time
points (Fig. 4B). A signicant increase in cell migration
was observed at 4, 12 and 24 h. IL-8, which was secreted
in signicant amounts by M2 macrophages, has been
shown to induce cancer cell migration.25 Furthermore,
macrophages are a major source of lipid mediators.9,11
Our previous data also indicate that CysLT1R signalling
is involved in intestinal epithelial cell proliferation, survival and migration.26 To determine which factors contribute to this increase in cell migration, SW480 cells
were pre-treated with a blocking antibody against the
IL-8 receptor CXCR1, the CysLT1R antagonist
ZM198,615 (the receptor for LTD4), or the CD47 blocking antibody B6H12. All of these treatments signi-

cantly blocked the cell migration mediated by the M2


macrophage-conditioned medium (Fig. 4C). After 6 or
24 h of stimulation with IL-8, LTD4, and M2 macrophage-conditioned medium, CD47 expression was signicantly increased in SW480 cells (Fig. 4D). These
ndings suggest that M2 macrophage-secreted IL-8
and LTD4, which is the ligand to CysLT1R, induces
colon cancer cell migration. CD47 signalling may be
involved in this induction.

3.6. CD47 and SIRPa expression in human colon cancer


tissue
The fact that CD47 inuences cell migration19,20 led
us to investigate CD47 expression in colon cancer tissue.
CD47 staining revealed that the tumour tissues contained higher levels of CD47 than the normal tissue
(Fig. 5A). In addition, the mRNA levels of CD47 were
signicantly higher (1.9-fold) in the tumour tissues compared to the normal tissues (Fig. 5B). In normal tissues,
SIRPa+ cells were found mostly below the epithelial
layer, whereas in tumour tissues, a signicant number
of SIRPa+ cells were observed between the epithelial
colon cancer cells (Fig. 5C); however, the mRNA

Fig. 5. CD47 and signal-regulatory protein a (SIRPa) expression in colon cancer tissues. (A) Immunohistochemistry and (B) mRNA expression of
CD47 in normal and tumour tissues from colon cancer patients. (C) Immunohistochemistry and (D) mRNA expression of SIRPa in normal and
tumour tissues from colon cancer patients. **P < 0.01 (column statistics).

Please cite this article in press as: Zhang Y. et al., Crosstalk between colon cancer cells and macrophages via inammatory mediators and CD47
promotes tumour cell migration, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.005

Y. Zhang et al. / European Journal of Cancer xxx (2013) xxxxxx

expression of the CD47 ligand SIRPa was similar in


normal and colon cancer tissues (Fig. 5D).
3.7. CD47 and SIRPa contribute to cell migration in
SW480 and M2 macrophage co-culture
As we have shown (Figs. 5 and 3A), colon cancer tissues expressed high levels of CD47, and M2 macrophages expressed high levels of SIRPa. Direct binding
of CD47 and SIRPa may contribute to cell migration.27
To investigate the role of the CD47SIRPa interaction
in cell migration, we used ibidi inserts to create a co-culture system (Fig. 6A). More pronounced cell migration
of SW480 cancer cells was observed under conditions
of co-culture with M2 macrophages (Fig. 6A). Under
co-culture conditions, SW480 cells and M2 macrophages migrated towards each other, resulting in cellto-cell contact after 24 h (Fig. 6B). These ndings
suggest that M2 macrophages and SW480 cells attract
each other and promote cellular migration. We next
pre-treated SW480 cells with the CD47 blocking antibody B6H12, and this treatment signicantly reduced
the SW480 and M2 macrophage migration (Fig. 6C).
Furthermore, following siRNA-induced downregulation
of CD47 in SW480 colon cancer cells (Fig. 6D), these
cancer cells exhibited signicantly reduced migration in
the presence of M2 macrophages compared to SW480
cells transfected with scrambled siRNA. This eect
was even further reduced in the presence of M2 macrophages pre-treated with SIRPa blocking antibodies
(Fig. 6E). Taken together, these ndings suggest that
an interaction between CD47 and SIRPa is involved in
the regulation of SW480 cancer cells and M2 macrophages migration under co-culture conditions.
4. Discussion
Macrophages constitute a major population of
tumour-inltrating immune cells that reside in the
tumour microenvironment.28 Depending on their phenotype, these cells exhibit either pro- or anti-tumour
properties. Many dierent types of cancer tissues are
populated by signicant numbers of CD206-positive
macrophages, and these cells are commonly referred to
as TAMs.29 In this study, we addressed the interplay
between TAMs, the inammatory microenvironment
and tumour cells in the context of colorectal cancer.
We observed a high number of macrophages, which
were immunohistochemically dened as CD68+ cells,
in the tumour microenvironment of tissue samples
obtained from colorectal cancer patients. TAMs have
an M2 macrophage-like phenotype,6 but since CD68 is
expressed on both M1 and M2 macrophages, CD68
expression alone does not dene TAMs. A number of
other and more specic markers, such as CD83, CD80
and CCR7 for M1 macrophages30,31 or CD206 and

11

CD16332 for M2 macrophages, have been identied.33


Compared to normal tissues, the number of CD68+
and CD206+ cells was signicantly higher in the colon
cancer microenvironment, especially in tissues from
Dukes C group of patients, compared to normal tissues.
The location of these CD68+ and CD206+ cells diered
between normal and tumour tissues. In tumour tissues,
these cells were mostly observed between tumour cells.
In this tumour microenvironment created by colon cancer cells and macrophages, macrophages may be
induced to a more tumour-promoting phenotype via
interaction of the cancer cells with TAMs, and this binding may be direct. We also observed a tendency of
increased CD206+ cells to be associated with a poorer
prognosis. Together, these ndings suggested that M2
macrophages are tumour-associated cells and that these
cells are involved in cancer progression. To further
address the importance of macrophages for colon cancer
development we also performed experiments with a
xenograft mouse model. Interestingly and in agreement
with our previous results, we observed higher macrophage content in larger tumours than in smaller tumours
in this model. It is quite plausible that our continuous
work will take advantage of a colour-coded uorescence
imaging model that can be used to study interactions
between human tumour cells and host cells in nude
mice.34,35 Homan and co-workers have recently developed this imaging method further to a tri-colour based
imaging method that can be used to visualise interactions between three dierent types of cells for example
that between host macrophages and lymphocytes with
human cancer cells in nude mice.36
To explore the interaction between M2 macrophages
and tumour cells, we used an in vitro model system.
Macrophages can be dierentiated into M1- or M2-like
macrophages in response to specic stimuli, and thereafter, these cells produce Th1 or Th2 cytokines.37 Using
in vitro dierentiated macrophages, we observed a significant increase in the secretion of Th1 cytokines, such as
IL-1b, IFN-c and TNF-a, by the dierentiated M1 macrophages, although the conditioned medium of M2 macrophages contained low but clearly detectable levels of
both IL-1b and TNF-a. Aside from controlling tumour
angiogenesis and invasiveness,38 IL-1b also activates the
Wnt signalling pathway in colon cancer cells, thereby
promoting proliferation.39 In addition to activating
macrophages, TNF-a has also been shown to promote
angiogenesis in the tumour environment.40,41 On the
other hand, in the conditioned medium of M2-dierentiated macrophages, the levels of many cytokines were
increased. Importantly, the Th2 cytokines IL-4 and
IL-10 were signicantly increased, and these cytokines
are known to be immunosuppressive and contribute to
M2 macrophage dierentiation.37,42 Interestingly, in this
context, we found that SW480 colon cancer cells secrete
very high levels of IL-10. Recently, Tanikawa and

Please cite this article in press as: Zhang Y. et al., Crosstalk between colon cancer cells and macrophages via inammatory mediators and CD47
promotes tumour cell migration, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.005

12

Y. Zhang et al. / European Journal of Cancer xxx (2013) xxxxxx

Fig. 6. Cell migration of SW480 colon cancer cells and M2 macrophages under co-cultured conditions. (A) Wound healing assay with ibidi inserts
shows cell migration between M2 macrophages and SW480 cells at dierent time points. (B) Representative immunouorescence images show
expression and localisation of CD47 (green) and signal-regulatory protein a (SIRPa) (red) in SW480 cells and M2-macrophages at 0 and 24 h in a
wound healing assay with ibidi inserts. DAPI is shown in blue. Scale bar is 20 lm. (C) Cell migration between M2 macrophages and SW480 cells after
treatment of SW480 cells with CD47 blocking antibody B6H12. (D) CD47 expression in SW480 cells after transfection with CD47 siRNA (siRNA 13)
or control scramble siRNA (scr). (E) Wound healing assay with ibidi inserts shows cell migration between SW480 cells transfected with CD47 siRNA
(siRNA 2 + 3) or control scramble siRNA (scr) and M2-like macrophages treated with SIRPa blocking antibody. (F) Schematic depicting the crosstalk
between SW480 colon cancer cells and M2 macrophages. *P < 0.05, **P < 0.01 (column statistics for D, two-tailed Students t-test for A), #P < 0.05
(two-way ANOVA). (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: Zhang Y. et al., Crosstalk between colon cancer cells and macrophages via inammatory mediators and CD47
promotes tumour cell migration, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.005

Y. Zhang et al. / European Journal of Cancer xxx (2013) xxxxxx

coworkers demonstrated the importance of IL-10 in


tumour development, growth and metastasis.43 We continued this line of investigation by studying the eect of
IL-10 on CD206 and SIRPa expression on M1-like macrophages. Indeed, IL-10 increased SIRPa expression in
M1-like macrophages. Furthermore, we found that the
expression pattern of CD206 was similar in M1-like
macrophages stimulated with IL-10 or co-cultured with
SW480 cells, which secrete IL-10. These ndings support
the notion that IL-10, which is produced by colon cancer cells and abundant in the tumour microenvironment,
contributes to M2 macrophage dierentiation.
Another very important cytokine, which is secreted
by M2 macrophages at a signicant level, is IL-8, which
has two receptors, CXCR1 and CXCR2.44 Functional
CXCR1 and CXCR2 mediate IL-8-triggered Ca2+
release, contraction and migration.45 In this study,
IL-8 was detected in M2 macrophage-conditioned medium
and was found to induce colon cancer cell migration
through its receptor CXCR1. IL-8 also inuenced the
expression of CD47, which is involved in colon cancer
cell migration. These results describe a novel role of
IL-8 in regulation of colon cancer cell migration.
Macrophages are also a major source of lipid mediators.9,11 In our study, a specic CysLT1R antagonist signicantly reduced cell migration after M2 macrophageconditioned medium stimulation of SW480 cells. This
nding is consistent with previous studies showing CysLT1R signalling is involved in intestinal epithelial cell
proliferation, survival and migration.26 Taken together,
these data highlight the crosstalk between macrophages
and colon cancer cells and indicate that it is mediated, at
least in part, by inammatory lipid mediators generated
from the tumour microenvironment.
Cancer cells interact with the microenvironment via a
number of adhesion molecules. In the present study, a
CD47 blocking antibody and CD47 siRNA signicantly
reduced cell migration in an M2 macrophage/SW480
colon cancer cell co-culture system. We also found that
the addition of a SIRPa blocking antibody to the above
co-culture system further reduced cell migration. This
nding is in accordance with our previous study in
which we reported that CD47 signalling participates in
the regulation of cyclooxygenase-2 (COX-2) expression
and thus, in triggering intestinal epithelial cell migration.20 We also found that CD47 expression was
increased at both the mRNA and protein levels in tissue
biopsies from colon cancer patients. Previous reports
indicated that CD47 is upregulated on tumour cells,
where it interacts with SIRPa expressed on macrophages
and allows tumour cells to evade macrophage clearance.17,46 Our ndings now identify an additional activity of CD47 in promoting cancer progression. The
interaction of CD47 and SIRPa is localised to the membrane distal domain of SIRPa.47 In agreement with such
a CD47SIRPa interaction, we observed that SIRP+

13

cells in tumours were mostly located between tumour


cells, and colon cancer cells SW480 interact with M2
macrophages under co-culture conditions, clearly supporting a physical interaction between CD47 and
SIRPa. In comparison to M1-dierentiated macrophages, SIRPa expression was signicantly higher in
M2-dierentiated macrophages.
Chao and co-workers have previously reported an
anti-tumour eect by administration of a blocking
anti-CD47 antibody in an animal model of acute lymphoblastic leukaemia (ALL).48 The authors proposed
that the observed eect of the anti-CD47 antibody was
due to blockage of the anti-phagocytotic eect of
CD47 enabling phagocytosis of ALL cells by macrophages. Furthermore, in a more recent study from the
same group, also commented by Spaargaren,49 it was
shown that anti-CD47 antibody treatment also prevent
the dissemination of lymphoma cells.50 Dierent possible explanations for this eect are presented or proposed. These include an eect on the anti-CD47
antibody on lymphoma cell migration and an increased
macrophage phagocytosis of circulating lymphoma
cells. Our data suggest that SIRPa expressed on macrophages promotes colon cancer progression by increasing
the migratory capacity of colon cancer cells via interaction with CD47 expressed on the tumour cells which is
in part in line with the suggested eect of CD47 in lymphoma cell dissemination.
In conclusion, as a major inammatory component
of the tumour microenvironment, TAMs promote colon
cancer cell migration via secretion of soluble mediators
and a possible cellcell interaction between CD47 on
cancer cells and its ligand SIRPa on macrophages
(Fig. 6F). Our ndings reveal important mechanisms
whereby the TAM-enriched tumour microenvironment
promotes colon cancer cell migration and subsequent
metastasis. Targeting these interactions with small
antagonists or blocking antibodies may constitute new
therapies for patients with colon cancer.
Conict of interest statement
None declared.
Acknowledgements
We thank P.A. Oldenborg from Umea University for
kindly providing CD47 antibody, R. Ehrnstrom for expert help with the pathological evaluations and E. Nilsson for invaluable technical assistance. This work was
supported by grants to A.S. from the Swedish Cancer
Foundation, the Swedish Medical Research Council,
the Foundations at Skanes University Hospital, Gunnar
sterlund Foundation,
Nilsson Foundation, and the O
and to W.S. and Y.Z. from the Royal Physiographical
Society in Lund.

Please cite this article in press as: Zhang Y. et al., Crosstalk between colon cancer cells and macrophages via inammatory mediators and CD47
promotes tumour cell migration, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.005

14

Y. Zhang et al. / European Journal of Cancer xxx (2013) xxxxxx

Appendix A. Supplementary data


Supplementary data associated with this article can
be found, in the online version, at http://dx.doi.org/
10.1016/j.ejca.2013.06.005.
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