Cvia inammatory mmigration

Yuan Zhang, Wondosse

Division of Cell and Experimental Patho

Sk 5 02 M

Colon cancerTumour-associated

Cell migration

stroma of many tumours and are frequently associated with the progression of several typesof cancer. We investigated the role of M2 macrophages in colon cancer progression and found

and CD206 (M2 macrophage marker) cells and increased CD47 expression. To explorepotential interplay between colon cancer cells and M2 macrophages, we differentiated the

SW480-conditioned medium. Similarly, M2 macrophage-conditioned medium inducedSW480 cell migration and CD47 expression. Factors released by macrophages were involved

migration of SW480 cells in the presence of M2 macrophages. This effect was further

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

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).

European Journal of Cancer (2013) xxx, xxxxxx

Ava i l ab l e a t www.sc i enced i r e c t . com

journa l homepage : www.e j cancer . comdecreased via blocking antibodies against the CD47 ligand signal-regulatory protein a(SIRPa). Additionally, cancer cells also secreted signicant levels of IL-10, thereby promotingM2 macrophage differentiation. These ndings indicate that a TAM-enriched tumour micro-environment promotes colon cancer cell migration and metastasis. 2013 Elsevier Ltd. All rights reserved.in this induction. In addition, SW480 cells migrated faster when co-cultured with M2 macro-phages. Inhibition of CD47 with blocking antibodies or siRNA signicantly reduced themonocyte cell line THP-1 into M1 and M2 macrophages (CD206high and Th2 cytokine-secret-ing cells), respectively. M2 macrophages migrated faster than M1 macrophages towardsmacrophageCD47

that human colon cancer tissue had elevated numbers of CD68+ (macrophage marker) cells+Ppane University Hospital, SE-20

KEYWORDSlease cite this article in pressromotes tumour cell migratioAas: Zhangn, Eur Jcolon cancer cells and macrophagesediators and CD47 promotes tumour cell

n Sime, Maria Juhas, Anita Sjolander

logy, Department of Laboratory Medicine, Clinical Research Centre, Lund University,

almo, Sweden

bstract Tumour-associated macrophages (TAMs) of the M2 phenotype are present in therosstalk betweenY. et al., Crosstalk between colon cancer cells and macrophages via inammatory mediators and CD47Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.005

1. Introduction contribute to cell migration. For example, CD47 has

2 Y. Zhang et al. / European Journal of Cancer xxx (2013) xxxxxxColorectal cancer is the third most commonly diag-nosed cancer in the world. Patients suering from inam-matory bowel diseases, such as Crohns disease andulcerative colitis, have an elevated risk of developingcolon cancer.1 In recent years, the tumour microenviron-ment has been highlighted as an important hallmark ofcancer.2,3 In tumour microenvironments, macrophagesrepresent amajor inammatory component of the stromaand aectmany aspects of the neoplastic tissue.4 Tumour-associated macrophages (TAMs) play an important rolein cancer progression, and accordingly, high levels ofmacrophage inltration of the tumour tissues are associ-ated with poor prognosis in cancer patients.4 Intratumo-ural macrophages that express the macrophage markersCD68 and CD163 are enriched in colon carcinoma andmay promote tumourigenesis5; however, the mechanismof such promotion remains unclear. Evidence indicatesthat intratumoural macrophages are recruited by tumourcells.6 The tumour microenvironment is thought toinduce the tumour-promoting M2 macrophage pheno-type.7 Therefore, specic visualisation of M2 macro-phages in colon cancer tissues is crucial.8

Macrophages produce a wide array of cytokines andlipid mediators, such as prostaglandins and leukotrienes(LTs).9 M1 macrophages are pro-inammatory andpotentiate Th1 responses by secreting high levels ofinterleukin (IL)-1, IL-12, interferon (IFN)-c and otherTh1 cytokines, whereas M2 macrophages secrete highlevels of IL-8 and Th2 cytokines, such as IL-4, IL-10and IL-13, which contribute to the maintenance of animmunosuppressive microenvironment.10 It has beendocumented that the prostaglandin E2 increases migra-tion and invasion of colon cancer cells.11 The pro-inammatory cysteinyl leukotriene D4 (LTD4) has alsobeen implicated in colon cancer progression,12 and wehave shown previously that LTD4 increases prolifera-tion, migration and survival in colon cancer cells viaCysLT1 receptor (CysLT1R) signals.

1315 Equallyimportant, we have shown that colon cancer patientswith high CysLT1R expression in their tumour tissueshave a poor prognosis.16 Therefore, we hypothesise thatLTD4 released from M2 macrophages inuences coloncancer cell behaviour.

Apart from soluble factors, cell surface-bound mole-cules regulate the interaction between intratumouralmacrophages and colon cancer cells. One such moleculethat was recently shown to be commonly expressed oncancer cells is CD47.17 CD47 was rst described as ananti-phagocytosis molecule based on its interaction withsignal regulatory protein alpha (SIRPa), which isexpressed on the surface of macrophages.18 In the con-text of cancer, such a mechanism could enable cancercells to escape the immune system.17 The interactionbetween CD47 and SIRPa has also been suggested toPlease cite this article in press as: Zhang Y. et al., Crosstalk between colonpromotes tumour cell migration, Eur J Cancer (2013), http://dx.doi.org/10been shown to control the migration of canine kidneyepithelial cells19 and intestinal epithelial cells grown ona collagen I-coated surface.20 Inhibition of CD47SIRPa interactions with a CD47 blocking antibodymay be a potential therapeutic approach17; however,the precise role of CD47 in colon cancer cell migrationand metastasis remains to be determined.

In this study, we report that M2-like macrophagessecrete immunosuppressive cytokines to create a micro-environment that promotes colon cancer cell growth andmigration. In addition, colon cancer cells exhibit ele-vated CD47 levels, which can be inuenced by macro-phages, and secrete high levels of IL-10, enhancing theM2 macrophage phenotype. Increased CD47SIRPasignalling between colon cancer cells and M2-like mac-rophages promotes cancer cell migration. These ndingshighlight the importance of TAM activity in the coloncancer microenvironment.

2. Materials and methods

2.1. Cell culture, stimulation and dierentiation

The human colon cancer cell line SW480,21 HCT-116and human monocyte cell line THP-1 (obtained fromATCC) were cultured following the suppliers instruc-tions. SW480 cells were stimulated with 20 ng/ml IL-4,80 nM LTD4, 100 ng/ml IL-8 or M2 macrophage-condi-tioned medium for 24 h at 37 C after overnight serumstarvation. THP-1 cells were dierentiated by treatmentwith 100 nM phorbol-12-myristate-13-acetate for 7days. The adhered cells were further dierentiated with100 ng/ml lipopolysaccharide for 72 h and 20 ng/mlIFN-c during the last 48 h (M1) or 20 ng/ml IL-4 for48 h (M2; Fig. 2A). For multiplex assays, enzyme-linkedimmunosorbent assays (ELISAs), and conditioned med-ium collecting, the dierentiated macrophages werewashed with serum-free medium to remove the dieren-tiation 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 cancerand control colon specimens from colorectal cancerpatients were obtained from the archives of the Depart-ment of Pathology at Malmo University Hospital.16,21

Tissues from 72 patients with varying grades and stagesof disease were included. Staging of the tumours wasdone using Dukes classication.22 Fresh biopsies wereobtained for mRNA analysis. The biopsy samples wereplaced in RNAlater (Qiagen, Hilden, Germany) and fro-zen by submersion in liquid nitrogen. The matched con-trol samples from normal colon tissues were surgicalspecimens from the same patients. Specimens werecancer cells and macrophages via inammatory mediators and CD47.1016/j.ejca.2013.06.005

Fig. 1. CD68 and CD206 expression in colon cancer tissues. (A) mRNA expression of CD68 and CD206 in normal and tumour tissues from coloncancer patients. (B and E) CD68 and CD206 immunohistochemistry staining. (C, D, F, and G) Number of CD68+ and CD206+ cells in tissues fromcolon cancer patients. (H) KaplanMeier survival curve using log-rank test. Numbers of CD206+ cells in tumour tissues were compared to normaltissues. 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).

Y. Zhang et al. / European Journal of Cancer xxx (2013) xxxxxx 3

Please cite this article in press as: Zhang Y. et al., Crosstalk between colon cancer cells and macrophages via inammatory mediators and CD47promotes tumour cell migration, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.005

Fig. 2. Dierentiation and cytokine proles of M1 and M2 macrophages derived from the human monocyte cell line THP-1. (A) Schematicdiagram 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 andCD206 in M1- and M2-like macrophages. (D) Signal-regulatory protein a (SIRPa) expression in M1- and M2-like macrophages. (E) Cytokineproles 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 areshown 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 Band D, two-tailed Students t-test for E).

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

Please cite this article in press as: Zhang Y. et al., Crosstalk between colon cancer cells and macrophages via inammatory mediators and CD47promotes tumour cell migration, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.005

obtained with informed consent after ethical approval 2.6. Immunouorescent staining

Y. Zhang et al. / European Journal of Cancer xxx (2013) xxxxxx 5was granted by the Ethics Committee of LundUniversity.

2.3. Tumour xenograft study

The Regional Ethics Committee for Animal Researchat Lund University, Sweden (M205-10) approved thepresent animal study. Female 6- to 8-week-old athymicnude mice (BalbCnu/nu) were purchased from TaconicEurope A/S (Ry, Denmark). To generate subcutaneoushuman colon cancer xenografts, 2.5 106 low-passageHCT-116 cells suspended in 100 ll phosphate-bueredsaline (PBS) were injected into both anks of the mice(21 mice in total). Twenty-one days after tumour cellinoculation all mice were sacriced, and the tumourswere removed. Tumour tissues were immediately xedin 10% buered formalin and then embedded in paranfor further immunohistochemical analysis.

2.4. Immunohistochemistry

Formalin-xed, paran-embedded archival coloncancer human specimens were stained with CD68(1:50; Biolegend, San Diego, CA, United States ofAmerica (USA)), CD206 (1:50; BD Biosciences, SanJose, 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 xeno-graphs were stained with the F4/80 (1:100; AbD Serotec,Raleigh, NC, USA) antibody. All stained tissues werevisualised by incubation with secondary peroxidase-con-jugated antibodies. After immunostaining, all slideswere manually counterstained with Mayers haematoxy-lin. Slides were scanned with the ScanScope CS (Aperio,Vista, CA, USA) at 10 and 40 magnication. Num-bers of CD68+ and CD206+ cells were counted withNIS-Elements Advanced Research software (Nikon,Tokyo, Japan) and statistically analysed with SPSS soft-ware. The total F4/80 positive stained area per tumourwas measured with Aeprio ImageScope software (Ape-rio, Vista, CA, USA).

2.5. RT-PCR

RNA from cells and tissue samples was isolatedfollowing the protocol of the Qiagen RNeasy PlusMini Kit.21 The following primers were obtainedfrom Applied Biosystems (Cambridge, UnitedKingdom (UK)): CD68 (Hs00154355_m1), CD206(Hs00267207_m1), CD47 (Hs00964717_m1), and SIRPa(Hs00388955_m1). Amplication was performed in aMx3005P system (Agilent Technologies, Inc., CA,USA), and reactions were analysed with MxPro soft-ware and normalised against the housekeeping geneHPRT1.Please cite this article in press as: Zhang Y. et al., Crosstalk between colonpromotes tumour cell migration, Eur J Cancer (2013), http://dx.doi.org/10THP-1 cells were attached to coverslips by centrifuga-tion at 1000 rpm for 5 min. Fixation and blocking wereperformed as previously described.20 The cells wereincubated with SIRPa, CD68, CD206 or mouse immu-noglobulin G (IgG) antibodies (1:100) for 1 h, washedwith PBS and incubated with Alexa 488 or Alexa 546goat anti-mouse secondary antibody (1:500) for 1 h.For double staining, an extra blocking step was per-formed. After washing with PBS, the cells were incu-bated with DAPI (1:1000) for 3 min and mounted inuorescent mounting medium (Dako, Glostrup, Den-mark). The slides were photographed with a NikonEclipse 80i microscope using a PlanApo 60 objectiveand NIS-Elements Advanced Research software(Fig. 2B), an Olympus FluoView FV10i ConfocalMicroscope using a 60 objective (Olympus Corpora-tion, Tokyo, Japan; Fig. 3B), or a Zeiss LSM 700 confo-cal microscope (Carl Zeiss Microscopy GmbH, Jena,Germany; Fig. 6A).

2.7. Flow cytometry

The dierentiated M1 and M2 macrophages weredetached using 0.02% versene and were washed twicein 0.5% bovine serum albumin (BSA)/PBS before block-ing the human FcRs with 20 lg/ml heat-aggregatedhuman IgG. Next, 0.5 105 cells were suspended in100 ll 0.5% BSA/PBS and incubated with 2 lg/mlCD68 antibody followed by incubation with Alexa 488goat anti-mouse secondary antibody (30 min each at4 C). Cells were incubated with 20 ll allophycocya-nin-conjugated CD206 (BD Biosciences) or 5 ll SIRPaantibody (eBioscience). Cells were examined with aFACS-Calibur using the software Cell Quest (BectonDickinson, San Jose, CA, USA) and analysed usingFCS Express Version 4 (De Novo Software, Los Ange-les, CA, USA). Forward and side scatter gates were setto include all viable cells.

2.8. Co-culture of M1 or M2 macrophages with SW480

cells

SW480 cells were counted before adding pre-dieren-tiated M1 or M2 macrophages at a ratio of 1:1 or 2:1and cultured in 1.5% FBS-containing medium for 72 h.The conditioned medium was collected for a multiplexassay or ELISA. Medium containing 1.5% FBS wasused as a negative control.

2.9. Measurement of secreted cytokines by multiplexassay

To remove cell debris, the conditioned mediumwas collected and centrifuged at 1000 rpm for 5 min.cancer cells and macrophages via inammatory mediators and CD47.1016/j.ejca.2013.06.005

6 Y. Zhang et al. / European Journal of Cancer xxx (2013) xxxxxxElectrochemiluminescence assays were performed onmacrophage-conditioned medium in duplicate using a10-plex human TH1/TH2 detection kit (Meso ScaleDiscovery, Gaithersburg, MD, USA) according to the

Fig. 3. Education of M1-like macrophages. (A) Histogram overlay showinmacrophages, M2 macrophages and M1 macrophages stimulated withimmunouorescence images showing expression and localisation of CD20IL-10 stimulation. (C) M1 macrophages co-cultured with SW480 cells. Dreferences to colour in this gure legend, the reader is referred to the web

Please cite this article in press as: Zhang Y. et al., Crosstalk between colonpromotes tumour cell migration, Eur J Cancer (2013), http://dx.doi.org/10manufacturers instructions. The plates were read usingthe Meso Scale Discovery SECTOR Imager 6000 andanalysed using Discovery Workbench and SoftMaxPRO 4.0 software.

g cell surface expression of signal-regulatory protein a (SIRPa) in M1IL-10 for 72 h and analysed by ow cytometry. (B) Representative6 (green) and SIRPa (red) in M2 and M1 macrophages with/withoutAPI is shown in blue. Scale bar is 20 lm. (For interpretation of theversion of this article.)

cancer cells and macrophages via inammatory mediators and CD47.1016/j.ejca.2013.06.005

Tween 20) or 5% non-fat dried milk. The membranes

20 lg/ml anti-CD47 antibody B6H1220 for 30 min,

tion of 0.02% versene and collected. Approximately

Cause of Death Registry and the Swedish Cancer Regis-

Y. Zhang et al. / European Journal of Cancer xxx (2013) xxxxxx 715 min or 20 min, respectively. The wound healing assaywas performed as described previously.20

For the co-cultured cell wound healing assay, M2macrophages were dierentiated in the wells of an ibidiculture-insert (ibidi, Martinsried, Germany). On day 4,2.5 106 SW480 cells were added to the dishes outsideof the insert. Thereafter, SW480 cells and macrophageswere cultured for an additional 5 days. On day 9, theculture medium from the macrophages was aspiratedbefore the insert was carefully removed. In some exper-iments, transfected SW480 cells were used and M2 mac-rophages in the ibidi wells were treated with 10 lg/mlanti-SIRPa antibody (Biolegend, San Diego, CA,USA) for 30 min. SW480-conditioned medium was col-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) anti-bodies. The membranes were washed and incubated for1 h at room temperature with HRP-conjugated second-ary antibodies diluted at 1:10,000. Proteins weredetected after incubation with an Immun-Star WesternChemiluminescence Kit (BioRad, Hercules, CA, USA)using a Bio-Rad ChemiDoc XRS+ System. Bio-RadImage Lab software was used for densitometric analysis,and the value obtained from the non-stimulated controlwas set to 100.

2.12. Wound healing assay

SW480 cells were serum starved for 2 h and incubatedwith or without 2 lg/ml IL-8 receptor CXCR1 antibody(Abcam, Cambridge, UK), 50 lM CysLT1R inhibitorZM198,615 (AstraZeneca, R&D Lund, Sweden), or2.10. Transfection with CD47 siRNA oligomers

SW480 cells were cultured for 3 days to 5060% con-uence. The growth medium was aspirated, and the cellswere washed with OPTI-MEM I (Invitrogen Corp.,Carlsbad, CA, USA). Next, 5 ml of OPTI medium con-taining 75 nM siRNA against CD47 (ID #: 2811,145978 and 145979) or a scrambled control siRNA(Ambion, Cambridgeshire, UK) was added to the cellsalong with lipofectamine 2000. After 4 h, the transfec-tion medium was diluted with RPMI 1640 medium(10% FBS) without antibiotics, and the cells wereallowed to grow for an additional 72 h with a mediumchange after 24 h.

2.11. Western blotting

Whole cell lysates were prepared, and Western blot-ting was performed as described.13 The polyvinylidenediuoride (PVDF) membranes were blocked for 1 h atroom temperature with either 4% BSA/PBS-T (0.05%Please cite this article in press as: Zhang Y. et al., Crosstalk between colonpromotes tumour cell migration, Eur J Cancer (2013), http://dx.doi.org/10Increased macrophage inltration has been detectedin many dierent solid tumours.23 To quantify and dis-tinguish the phenotype of these macrophages, expres-sion of CD68, which is expressed at high levels onmacrophages, and CD206, which is highly expressedon M2 macrophages, was examined by Q-PCR andcompared between normal and tumour tissues fromter. The overall survival was calculated as colon cancer-specic death. Deaths due to other causes were censoredat the time of death. Prizm software 5.0d (GraphPadSoftware, San Diego, CA, USA) was used for other sta-tistical analyses. All the data are presented as themean standard error of the mean (SEM), and statisti-cal signicance was determined as P < 0.05 by a two-way analysis of variance (ANOVA; labelled #), Columnstatistics or a two-tailed Students t-test (labelled ). Allmeans were calculated from data from at least threeindependent experiments.

3. Results

3.1. Macrophage content in human colon cancer tissue5 104 cells were added to the upper well of a Boydenchamber (Neuro Probe, Gaithersburg, MD, USA).The lower well contained supplemented RPMI 1640(10% FBS) or SW480-conditioned medium. The twowells were separated by an 8.0-lm polycarbonate PVDFmembrane. After a 4-h incubation at 37 C, cells thathad not migrated were removed, and the membranewith migrated cells was xed and stained as describedpreviously.20 The membrane was washed, the remainingdye solubilised in 10% sodium dodecyl sulphate and theabsorbance measured at 590 nm.

2.14. Statistical analyses

SPSS software 16.0 was used for all immunostaininganalyses. Univariate survival analysis was performed byKaplanMeier analysis with a log-rank test to determinethe risk of death. Survival time was measured from thedate of surgery to the date of death or 80 months of fol-low-up. The death information is from the Swedishlected, centrifuged to remove cell debris and added backto the dishes. Non-adherent cells were gently washed inPBS. The cells were allowed to migrate for 24 h at 37 C.Pictures were taken20 with a Nikon DS-Fi1 microscopeusing a 10 objective and analysed with NIS-ElementsBasic Research software. The area of the wound wasmeasured with Adobe Photoshop CS4 software.

2.13. Boyden chamber assay

M1 and M2 macrophages were detached by the addi-cancer cells and macrophages via inammatory mediators and CD47.1016/j.ejca.2013.06.005

colon cancer patients. Expression of CD206 was 1.8-fold

array with CD206 antibody revealed signicantly

8 Y. Zhang et al. / European Journal of Cancer xxx (2013) xxxxxxincreased numbers of CD206+ cells in the tumour tissuesas compared with normal tissues (Fig. 1F and G), andthese cells were located between the epithelial cancercells (Fig. 1E). This nding indicated that the tumourarea contained more M2 macrophages than the normalarea. Additionally, we observed that a higher number ofCD206+ cells in the tumour than in the normal tissuetends to confer a poorer prognosis (Fig. 1H).

To assess the above results from both the in vitroexperiments and human colon cancer tissue we nextset up a mouse xenograft-model with human colon can-cer cells. We found, in good agreement with our humanin vivo results, a higher content of macrophages (F4/80positive cells) in the larger tumours as comparedwith the smaller tumours (Appendix SupplementaryFig. S1). These results further stress the importance ofTAMs 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 monocytecell line THP-1 was dierentiated into M2 or M1 macro-phages (Fig. 2A). THP-1 cells that normally grow in sus-pension and have a rounded morphology becameattached 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 macro-phages, the mRNA levels of macrophage markers wereinvestigated. Both M1 and M2 macrophages expressedsimilar levels of CD68 mRNA, but the mRNA levelsof CD206 were signicantly higher (23-fold) in the M2population (Fig. 2B). A similar expression pattern formacrophage marker expression was observed at the pro-tein level using ow cytometry (Fig. 2C).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 normaltissues, the CD68+ cells were primarily located outsidethe epithelial cell layer, whereas in the tumour tissue,these cells were observed mostly between the epithelialcancer cells (Fig. 1B). The number of CD68+ cells wasdetermined using a colon cancer tissue array, which con-tains 72 pairs of normal mucosa and tumour tissuepatient samples. In this experiment, tissues from 62 ofthe 72 patients were grouped, according to Dukes stageclassication. Signicantly more macrophages wereobserved in tumour tissues compared with normal tis-sues (Fig. 1C), especially in the late stage of colon cancerclassied as Dukes C (Fig. 1D).

Similarly, staining of the same colon cancer tissuePlease cite this article in press as: Zhang Y. et al., Crosstalk between colonpromotes tumour cell migration, Eur J Cancer (2013), http://dx.doi.org/10lated M1 macrophages, similar to that observed in dif-ferentiated M2 macrophages (Fig. 3B). Expression ofSIRPa increased from a mean uorescent intensity of178 to 249 in M1 macrophages stimulated with IL-10for 72 h (Fig. 3A). This increase in SIRPa expressionwas conrmed by immunouorescence staining inIL-10-stimulated cells (Fig. 3B). Furthermore,phage-conditioned media (Fig. 2E). IL-4 secretion wasobserved in M2 but not M1 macrophage-conditionedmedia. IL-8 expression was dramatically increased inthe conditioned media from M2 macrophages co-cul-tured with SW480 cells than in media from M2 macro-phages alone (from 673 to 4886 pg/ml). Moreover, theTh1 cytokines, including IL-1b, IFN-c and tumournecrosis factor (TNF)-a, were more highly expressedin the M1 macrophage conditioned medium. Interest-ingly, the IL-10 concentration in the SW480-conditionedmedium (204 pg/ml) was much higher than that in themacrophage-conditioned medium.

The cytokine proles from the M1 and M2 macro-phages conrmed the macrophage dierentiation e-ciency, and these data highlighted several cytokinesknown to inuence the behaviour of the tumour micro-environment. IL-10 is an immunosuppressive cytokinethat is known to be important in the dierentiation ofM2 macrophages.24 Therefore, we investigated the roleof IL-10 in macrophage polarisation.

3.4. IL-10 secreted from SW480 cells polarise M1

macrophages

SW480 cancer cells secreted signicant amounts ofIL-10 (Fig. 2E). We therefore hypothesised that M1macrophages dierentiate into M2 macrophages in thecancer cell milieu. We stimulated dierentiated M1 mac-rophages with IL-10 for 72 h and followed changes inexpression of CD206 and SIRPa. Fluorescent stainingof CD206 was increased at the cell membrane of stimu-In addition, signicantly higher expression of SIRPawasobserved at both themRNA(2.5-fold) andprotein lev-els (3.8-fold) in the dierentiated M2 macrophages com-pared to the dierentiated M1 macrophages (Figs. 2Dand 3A). This result suggests that colon cancer cells thatexpress high levels of CD47 interact withM2macrophagesvia an interaction between CD47 and SIRPa.

3.3. Cytokine proles of THP-1-derived macrophages

Macrophages derived from various tissue sites areknown to exhibit dierent cytokine proles.10 Thus, wemeasured the levels of secreted cytokines in conditionedmedia from cells grown under dierent conditions asdescribed 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 macro-phage-conditioned medium compared to the M1 macro-cancer cells and macrophages via inammatory mediators and CD47.1016/j.ejca.2013.06.005

immunouorescent staining indicated that CD206 andSIRPa expression were increased in M1 macrophagesco-cultured with IL-10-releasing SW480 cancer cellsfor 72 h (Fig. 3C). These ndings support the hypothesisthat M1 macrophages are prone to become M2-likewhen exposed to factors, such as IL-10, that are releasedby 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 investi-gate the ability of SW480 colon cancer cells to attractmacrophages. Signicantly more M2 macrophages than

pham.d hentibcroe ex.

Y. Zhang et al. / European Journal of Cancer xxx (2013) xxxxxx 9Fig. 4. Migration of SW480 colon cancer cells stimulated by M2 macroand M2-like macrophages migrate towards SW480-conditioned mediumacrophage-conditioned medium at indicated time points. (C) Wounblocking antibody, CysLT1R inhibitor ZM198,615, or CD47 blocking a(D) CD47 expression in SW480 cells upon IL-4, IL-8, LTD4 or M2 mashown as the mean standard error of the mean (SEM) of 37 separattwo-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 colonpromotes tumour cell migration, Eur J Cancer (2013), http://dx.doi.org/10ge-derived factors. (A) Boyden chamber assay demonstrates that M1-(B) Wound healing assay involving SW480 cells with or without M2aling assay with SW480 cells pre-treated with IL-8 receptor CXCR1ody B6H12 and stimulated with M2 macrophage-conditioned medium.phage-conditioned medium stimulation after 6 or 24 h. The results areperiments. *P < 0.05, **P < 0.01, ***P < 0.001 (column statistics for D,cancer cells and macrophages via inammatory mediators and CD47.1016/j.ejca.2013.06.005

M1 macrophages migrated towards the SW480 cancercell-conditioned medium (Fig. 4A). The tumour micro-environment is rich with various soluble and non-solu-ble factors, which are believed to be essential fortumour progression. In this study, M2 macrophageswere shown to release cytokines, and inammatorymediators, and thus, we examined the contribution ofthese dierent factors to the interaction between M2macrophages and colon cancer cells as well as to cellmigration. In a wound healing assay, SW480 cells werestimulated with M2 macrophage-conditioned medium,and wound closure was measured at dierent timepoints (Fig. 4B). A signicant increase in cell migrationwas observed at 4, 12 and 24 h. IL-8, which was secretedin signicant amounts by M2 macrophages, has beenshown to induce cancer cell migration.25 Furthermore,macrophages are a major source of lipid mediators.9,11

Our previous data also indicate that CysLT1R signallingis involved in intestinal epithelial cell proliferation, sur-vival and migration.26 To determine which factors con-tribute to this increase in cell migration, SW480 cellswere pre-treated with a blocking antibody against theIL-8 receptor CXCR1, the CysLT1R antagonistZM198,615 (the receptor for LTD4), or the CD47 block-ing antibody B6H12. All of these treatments signi-

cantly blocked the cell migration mediated by the M2macrophage-conditioned medium (Fig. 4C). After 6 or24 h of stimulation with IL-8, LTD4, and M2 macro-phage-conditioned medium, CD47 expression was sig-nicantly increased in SW480 cells (Fig. 4D). Thesendings suggest that M2 macrophage-secreted IL-8and LTD4, which is the ligand to CysLT1R, inducescolon cancer cell migration. CD47 signalling may beinvolved in this induction.

3.6. CD47 and SIRPa expression in human colon cancertissue

The fact that CD47 inuences cell migration19,20 ledus to investigate CD47 expression in colon cancer tissue.CD47 staining revealed that the tumour tissues con-tained higher levels of CD47 than the normal tissue(Fig. 5A). In addition, the mRNA levels of CD47 weresignicantly higher (1.9-fold) in the tumour tissues com-pared to the normal tissues (Fig. 5B). In normal tissues,SIRPa+ cells were found mostly below the epitheliallayer, whereas in tumour tissues, a signicant numberof SIRPa+ cells were observed between the epithelialcolon cancer cells (Fig. 5C); however, the mRNA

n cmus).

10 Y. Zhang et al. / European Journal of Cancer xxx (2013) xxxxxxFig. 5. CD47 and signal-regulatory protein a (SIRPa) expression in coloCD47 in normal and tumour tissues from colon cancer patients. (C) Imtumour tissues from colon cancer patients. **P < 0.01 (column statisticPlease cite this article in press as: Zhang Y. et al., Crosstalk between colonpromotes tumour cell migration, Eur J Cancer (2013), http://dx.doi.org/10ancer tissues. (A) Immunohistochemistry and (B) mRNA expression ofnohistochemistry and (D) mRNA expression of SIRPa in normal andcancer cells and macrophages via inammatory mediators and CD47.1016/j.ejca.2013.06.005

expression of the CD47 ligand SIRPa was similar in CD16332 for M2 macrophages, have been identied.33

Y. Zhang et al. / European Journal of Cancer xxx (2013) xxxxxx 11normal and colon cancer tissues (Fig. 5D).

3.7. CD47 and SIRPa contribute to cell migration inSW480 and M2 macrophage co-culture

As we have shown (Figs. 5 and 3A), colon cancer tis-sues expressed high levels of CD47, and M2 macro-phages expressed high levels of SIRPa. Direct bindingof CD47 and SIRPa may contribute to cell migration.27

To investigate the role of the CD47SIRPa interactionin cell migration, we used ibidi inserts to create a co-cul-ture system (Fig. 6A). More pronounced cell migrationof SW480 cancer cells was observed under conditionsof co-culture with M2 macrophages (Fig. 6A). Underco-culture conditions, SW480 cells and M2 macro-phages migrated towards each other, resulting in cell-to-cell contact after 24 h (Fig. 6B). These ndingssuggest that M2 macrophages and SW480 cells attracteach other and promote cellular migration. We nextpre-treated SW480 cells with the CD47 blocking anti-body B6H12, and this treatment signicantly reducedthe SW480 and M2 macrophage migration (Fig. 6C).Furthermore, following siRNA-induced downregulationof CD47 in SW480 colon cancer cells (Fig. 6D), thesecancer cells exhibited signicantly reduced migration inthe presence of M2 macrophages compared to SW480cells transfected with scrambled siRNA. This eectwas even further reduced in the presence of M2 macro-phages pre-treated with SIRPa blocking antibodies(Fig. 6E). Taken together, these ndings suggest thatan interaction between CD47 and SIRPa is involved inthe regulation of SW480 cancer cells and M2 macro-phages migration under co-culture conditions.

4. Discussion

Macrophages constitute a major population oftumour-inltrating immune cells that reside in thetumour microenvironment.28 Depending on their phe-notype, these cells exhibit either pro- or anti-tumourproperties. Many dierent types of cancer tissues arepopulated by signicant numbers of CD206-positivemacrophages, and these cells are commonly referred toas TAMs.29 In this study, we addressed the interplaybetween TAMs, the inammatory microenvironmentand tumour cells in the context of colorectal cancer.

We observed a high number of macrophages, whichwere immunohistochemically dened as CD68+ cells,in the tumour microenvironment of tissue samplesobtained from colorectal cancer patients. TAMs havean M2 macrophage-like phenotype,6 but since CD68 isexpressed on both M1 and M2 macrophages, CD68expression alone does not dene TAMs. A number ofother and more specic markers, such as CD83, CD80and CCR7 for M1 macrophages30,31 or CD206 andPlease cite this article in press as: Zhang Y. et al., Crosstalk between colonpromotes tumour cell migration, Eur J Cancer (2013), http://dx.doi.org/10Compared to normal tissues, the number of CD68+

and CD206+ cells was signicantly higher in the coloncancer microenvironment, especially in tissues fromDukes C group of patients, compared to normal tissues.The location of these CD68+ and CD206+ cells dieredbetween normal and tumour tissues. In tumour tissues,these cells were mostly observed between tumour cells.In this tumour microenvironment created by colon can-cer cells and macrophages, macrophages may beinduced to a more tumour-promoting phenotype viainteraction of the cancer cells with TAMs, and this bind-ing may be direct. We also observed a tendency ofincreased CD206+ cells to be associated with a poorerprognosis. Together, these ndings suggested that M2macrophages are tumour-associated cells and that thesecells are involved in cancer progression. To furtheraddress the importance of macrophages for colon cancerdevelopment we also performed experiments with axenograft mouse model. Interestingly and in agreementwith our previous results, we observed higher macro-phage content in larger tumours than in smaller tumoursin this model. It is quite plausible that our continuouswork will take advantage of a colour-coded uorescenceimaging model that can be used to study interactionsbetween human tumour cells and host cells in nudemice.34,35 Homan and co-workers have recently devel-oped this imaging method further to a tri-colour basedimaging method that can be used to visualise interac-tions between three dierent types of cells for examplethat between host macrophages and lymphocytes withhuman cancer cells in nude mice.36

To explore the interaction between M2 macrophagesand tumour cells, we used an in vitro model system.Macrophages can be dierentiated into M1- or M2-likemacrophages in response to specic stimuli, and thereaf-ter, these cells produce Th1 or Th2 cytokines.37 Usingin vitro dierentiated macrophages, we observed a signif-icant increase in the secretion of Th1 cytokines, such asIL-1b, IFN-c and TNF-a, by the dierentiated M1 mac-rophages, although the conditioned medium of M2 mac-rophages contained low but clearly detectable levels ofboth IL-1b and TNF-a. Aside from controlling tumourangiogenesis and invasiveness,38 IL-1b also activates theWnt signalling pathway in colon cancer cells, therebypromoting proliferation.39 In addition to activatingmacrophages, TNF-a has also been shown to promoteangiogenesis in the tumour environment.40,41 On theother hand, in the conditioned medium of M2-dieren-tiated macrophages, the levels of many cytokines wereincreased. Importantly, the Th2 cytokines IL-4 andIL-10 were signicantly increased, and these cytokinesare known to be immunosuppressive and contribute toM2 macrophage dierentiation.37,42 Interestingly, in thiscontext, we found that SW480 colon cancer cells secretevery high levels of IL-10. Recently, Tanikawa andcancer cells and macrophages via inammatory mediators and CD47.1016/j.ejca.2013.06.005

Fig. 6. Cell migration of SW480 colon cancer cells and M2 macrophages under co-cultured conditions. (A) Wound healing assay with ibidi insertsshows cell migration between M2 macrophages and SW480 cells at dierent time points. (B) Representative immunouorescence images showexpression and localisation of CD47 (green) and signal-regulatory protein a (SIRPa) (red) in SW480 cells and M2-macrophages at 0 and 24 h in awound healing assay with ibidi inserts. DAPI is shown in blue. Scale bar is 20 lm. (C) Cell migration betweenM2macrophages and SW480 cells aftertreatment of SW480 cells withCD47blocking antibodyB6H12. (D)CD47 expression in SW480 cells after transfectionwithCD47 siRNA (siRNA13)or control scramble siRNA (scr). (E)Wound healing assay with ibidi inserts shows cell migration between SW480 cells transfected with CD47 siRNA(siRNA2 + 3) or control scramble siRNA(scr) andM2-likemacrophages treatedwithSIRPablocking antibody. (F) Schematic depicting the crosstalkbetween 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.)

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

Please cite this article in press as: Zhang Y. et al., Crosstalk between colon cancer cells and macrophages via inammatory mediators and CD47promotes tumour cell migration, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.005

coworkers demonstrated the importance of IL-10 in cells in tumours were mostly located between tumour

Y. Zhang et al. / European Journal of Cancer xxx (2013) xxxxxx 13tumour development, growth and metastasis.43 We con-tinued this line of investigation by studying the eect ofIL-10 on CD206 and SIRPa expression on M1-like mac-rophages. Indeed, IL-10 increased SIRPa expression inM1-like macrophages. Furthermore, we found that theexpression pattern of CD206 was similar in M1-likemacrophages stimulated with IL-10 or co-cultured withSW480 cells, which secrete IL-10. These ndings supportthe notion that IL-10, which is produced by colon can-cer cells and abundant in the tumour microenvironment,contributes to M2 macrophage dierentiation.

Another very important cytokine, which is secretedby M2 macrophages at a signicant level, is IL-8, whichhas two receptors, CXCR1 and CXCR2.44 FunctionalCXCR1 and CXCR2 mediate IL-8-triggered Ca2+

release, contraction and migration.45 In this study,IL-8 was detected in M2 macrophage-conditioned mediumand was found to induce colon cancer cell migrationthrough its receptor CXCR1. IL-8 also inuenced theexpression of CD47, which is involved in colon cancercell migration. These results describe a novel role ofIL-8 in regulation of colon cancer cell migration.

Macrophages are also a major source of lipid media-tors.9,11 In our study, a specic CysLT1R antagonist sig-nicantly reduced cell migration after M2 macrophage-conditioned medium stimulation of SW480 cells. Thisnding is consistent with previous studies showing Cys-LT1R signalling is involved in intestinal epithelial cellproliferation, survival and migration.26 Taken together,these data highlight the crosstalk between macrophagesand colon cancer cells and indicate that it is mediated, atleast in part, by inammatory lipid mediators generatedfrom the tumour microenvironment.

Cancer cells interact with the microenvironment via anumber of adhesion molecules. In the present study, aCD47 blocking antibody and CD47 siRNA signicantlyreduced cell migration in an M2 macrophage/SW480colon cancer cell co-culture system. We also found thatthe addition of a SIRPa blocking antibody to the aboveco-culture system further reduced cell migration. Thisnding is in accordance with our previous study inwhich we reported that CD47 signalling participates inthe regulation of cyclooxygenase-2 (COX-2) expressionand thus, in triggering intestinal epithelial cell migra-tion.20 We also found that CD47 expression wasincreased at both the mRNA and protein levels in tissuebiopsies from colon cancer patients. Previous reportsindicated that CD47 is upregulated on tumour cells,where it interacts with SIRPa expressed on macrophagesand allows tumour cells to evade macrophage clear-ance.17,46 Our ndings now identify an additional activ-ity of CD47 in promoting cancer progression. Theinteraction of CD47 and SIRPa is localised to the mem-brane distal domain of SIRPa.47 In agreement with sucha CD47SIRPa interaction, we observed that SIRP+Please cite this article in press as: Zhang Y. et al., Crosstalk between colonpromotes tumour cell migration, Eur J Cancer (2013), http://dx.doi.org/10cells, and colon cancer cells SW480 interact with M2macrophages under co-culture conditions, clearly sup-porting a physical interaction between CD47 andSIRPa. In comparison to M1-dierentiated macro-phages, SIRPa expression was signicantly higher inM2-dierentiated macrophages.

Chao and co-workers have previously reported ananti-tumour eect by administration of a blockinganti-CD47 antibody in an animal model of acute lym-phoblastic leukaemia (ALL).48 The authors proposedthat the observed eect of the anti-CD47 antibody wasdue to blockage of the anti-phagocytotic eect ofCD47 enabling phagocytosis of ALL cells by macro-phages. Furthermore, in a more recent study from thesame group, also commented by Spaargaren,49 it wasshown that anti-CD47 antibody treatment also preventthe dissemination of lymphoma cells.50 Dierent possi-ble explanations for this eect are presented or pro-posed. These include an eect on the anti-CD47antibody on lymphoma cell migration and an increasedmacrophage phagocytosis of circulating lymphomacells. Our data suggest that SIRPa expressed on macro-phages promotes colon cancer progression by increasingthe migratory capacity of colon cancer cells via interac-tion with CD47 expressed on the tumour cells which isin part in line with the suggested eect of CD47 in lym-phoma cell dissemination.

In conclusion, as a major inammatory componentof the tumour microenvironment, TAMs promote coloncancer cell migration via secretion of soluble mediatorsand a possible cellcell interaction between CD47 oncancer cells and its ligand SIRPa on macrophages(Fig. 6F). Our ndings reveal important mechanismswhereby the TAM-enriched tumour microenvironmentpromotes colon cancer cell migration and subsequentmetastasis. Targeting these interactions with smallantagonists or blocking antibodies may constitute newtherapies for patients with colon cancer.

Conict of interest statement

None declared.

Acknowledgements

We thank P.A. Oldenborg from Umea University forkindly providing CD47 antibody, R. Ehrnstrom for ex-pert help with the pathological evaluations and E. Nils-son for invaluable technical assistance. This work wassupported by grants to A.S. from the Swedish CancerFoundation, the Swedish Medical Research Council,the Foundations at Skanes University Hospital, GunnarNilsson Foundation, and the Osterlund Foundation,and to W.S. and Y.Z. from the Royal PhysiographicalSociety in Lund.cancer cells and macrophages via inammatory mediators and CD47.1016/j.ejca.2013.06.005

14 Y. Zhang et al. / European Journal of Cancer xxx (2013) xxxxxxAppendix A. Supplementary data

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

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Y. Zhang et al. / European Journal of Cancer xxx (2013) xxxxxx 15Please cite this article in press as: Zhang Y. et al., Crosstalk between colonpromotes tumour cell migration, Eur J Cancer (2013), http://dx.doi.org/10cancer cells and macrophages via inammatory mediators and CD47.1016/j.ejca.2013.06.005

Crosstalk between colon cancer cells and macrophages via inflammatory mediators and CD47 promotes tumour cell migration1 Introduction2 Materials and methods2.1 Cell culture, stimulation and differentiation2.2 Patient samples2.3 Tumour xenograft study2.4 Immunohistochemistry2.5 RT-PCR2.6 Immunofluorescent staining2.7 Flow cytometry2.8 Co-culture of M1 or M2 macrophages with SW480 cells2.9 Measurement of secreted cytokines by multiplex assay2.10 Transfection with CD47 siRNA oligomers2.11 Western blotting2.12 Wound healing assay2.13 Boyden chamber assay2.14 Statistical analyses

3 Results3.1 Macrophage content in human colon cancer tissue3.2 M1- and M2-like macrophages differentiated from the human monocyte cell line THP-13.3 Cytokine profiles of THP-1-derived macrophages3.4 IL-10 secreted from SW480 cells polarise M1 macrophages3.5 Migration of SW480 colon cancer cells is induced by M2 macrophage-derived factors3.6 CD47 and SIRP expression in human colon can3.7 CD47 and SIRP contribute to cell migration

4 DiscussionConflict of interest statementAcknowledgementsAppendix A Supplementary dataAppendix A Supplementary dataReferences