bone marrow derived cd11bþjagged2þ cells promote...

Microenvironment and Immunology

Bone Marrow–Derived CD11bþJagged2þ Cells PromoteEpithelial-to-Mesenchymal Transition andMetastasization inColorectal Cancer

Francisco Caiado1,4, Tania Carvalho1,4, Isadora Rosa2, Leonor Rem�edio1, Ana Costa1, Jo~ao Matos3,Beate Heissig6, Hideo Yagita8, Koichi Hattori7, Jo~ao Pereira da Silva2, Paulo Fidalgo2, Ant�onio Dias Pereira2,and S�ergio Dias1,4,5

AbstractTimely detection of colorectal cancer metastases may permit improvements in their clinical management.

Here, we investigated a putative role for bone marrow–derived cells in the induction of epithelial-to-mesenchymaltransition (EMT) as a marker for onset of metastasis. In ectopic and orthotopic mouse models of colorectal cancer,bone marrow–derived CD11b(Itgam)þJagged2 (Jag2)þ cells infiltrated primary tumors and surrounded tumorcells that exhibited diminished expression of E-cadherin and increased expression of vimentin, 2 hallmarks of EMT.In vitro coculture experiments showed that the bone marrow–derived CD11bþJag2þ cells induced EMT through aNotch-dependent pathway. Using neutralizing antibodies, we imposed a blockade on CD11bþ cells' recruitment totumors, which decreased the tumor-infiltrating CD11bþJag2þ cell population of interest, decreasing tumor growth,restoring E-cadherin expression, and delaying EMT. In support of these results, we found that peripheral bloodlevels of CD11bþJag2þ cells in mouse models of colorectal cancer and in a cohort of untreated patients withcolorectal cancer were indicative of metastatic disease. In patients with colorectal cancer, the presence ofcirculating CD11bþJag2þ cells was accompanied by loss of E-cadherin in the corresponding patient tumors. Takentogether, our results show that bone marrow–derived CD11bþJag2þ cells, which infiltrate primary colorectaltumors, are sufficient to induce EMT in tumor cells, thereby triggering onset ofmetastasis. Furthermore, they arguethat quantifying circulating CD11bþJag2þ cells in patients may offer an indicator of colorectal cancer progressionto metastatic levels of the disease. Cancer Res; 73(14); 4233–46. �2013 AACR.

IntroductionMetastatic disease is a major cause of cancer-associated

mortality. Despite significant advances in the treatment ofprimary tumors, metastases remain a significant clinicalproblem, likely reflecting our limited knowledge of themechanisms governing this complex process (1). It is accept-

ed that metastasization follows a series of interrelated steps,each of which can be rate-limiting. These steps include: localinvasion by tumor cells, entry into systemic circulation(intravasation), invasion of the target organ (extravasation),and finally proliferation and growth of the secondary tumor(2). One of the major processes regulating local invasion inepithelial tumors is termed epithelial-to-mesenchymal tran-sition (EMT; refs. 3, 4). EMT is a transcriptional regulatedtransdifferentiation process characterized at the tumor celllevel, by a decrease in epithelial markers such as E-cadherin,loss of cell–cell adhesion, apical–basal polarity, and acqui-sition of mesenchymal markers such as vimentin associatedwith an increase in cell motility and invasion capacity (5–8).EMT has been positively correlated with increase breast andcolon cancer metastasis and decreased patient survival(3, 9, 10).

In the last decade there has been increasing evidencesuggesting that tumor metastasis is also regulated by nonma-lignant cells of the tumor microenvironment, namely by bonemarrow–derived cell populations (11). In fact distinct bonemarrow–derived populations such as tumor-associatedmacrophages (12, 13), premetastatic niche cells (14), andendothelial progenitor cells (15) have been shown to enhancemetastasization via multiple processes. Nevertheless, a directrole of bone marrow–derived cells in promoting EMT at the

Authors' Affiliations: 1Angiogenesis Lab, CIPM, 2Department of Gastro-enterology, and 3Histopathology Unit, Portuguese Institute of Oncology,IPOLFG, EPE; 4Instituto deMedicinaMolecular, Faculdade deMedicina deLisboa, Lisbon; and 5Instituto Gulbenkian de Ciência, Oeiras, Portugal;6Division of Stem Cell Dynamics and 7Laboratory of Stem Cell Regulation,Center for Stem Cell Biology & Regenerative Medicine, Institute of MedicalScience, University of Tokyo; and 8Department of Immunology, JuntendoUniversity School of Medicine, Tokyo, Japan

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Current address for S. Dias: Instituto de Medicina Molecular, Edificio EgasMoniz, Faculdade de Medicina da Universidade de Lisboa, 1649-028Lisboa, Portugal.

Corresponding Author: S�ergio Dias, Instituto de Medicina Molecular,Edificio Egas Moniz, Faculdade de Medicina da Universidade de Lisboa,1649-028 Lisboa, Portugal. Phone: 351-21-7999449; Fax: 351-21-7999411; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-0085

�2013 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 4233

on November 8, 2018. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 30, 2013; DOI: 10.1158/0008-5472.CAN-13-0085

http://cancerres.aacrjournals.org/

primary tumor has not been described, and was the focus ofthe present study.

Using ectopic and orthotopic colorectal cancer models inmice, we show that a population of bone marrow–derivedmyeloid (CD11bþF4/80þ) expressing Jagged2 (Jag2) is activelyrecruited into colon tumors and accumulates in tumor areasundergoing EMT. Detailed analysis of this tumor: bone mar-row–derived cell interaction shows the latter induce EMT viaNotch activation on the tumor cells. Importantly, in vivodepletion of CD11bþ cells in ectopic colorectal cancer modelsreduced the recruitment of CD11bþJag2þ cells into the tumorsand significantly decreased EMT. Quantification of circulating(peripheral blood) and tumor-derived CD11bþJag2þ cells inpatients with colorectal cancer was significantly correlatedwith the presence of metastatic disease. Together, the datapresented here reveal a novel undisclosed role for bone mar-row–derived cells in inducingEMT inprimary colorectal cancerand identifies a bonemarrow–derived cell population thatmaybe targeted and studied as a biomarker for colorectal cancermetastases formation.

Materials and MethodsHuman peripheral blood samples collection andprocessing

Peripheral blood samples of patients with sequential colo-rectal cancer evaluated at diagnosis by the MultidisciplinaryColorectal Cancer Team were collected at the Gastroenterol-ogy Department at Instituto Portugues de Oncologia (IPO,Lisbon, Portugal) after informed consent and InstitutionalReview Board approval (IPO), in accordance with the Decla-ration of Helsinki. Patients were included if they had a pathol-ogy exam showing colorectal adenocarcinoma. All patientspreviously submitted to endoscopic, surgical, or medical treat-ment for colorectal cancer were excluded. Peripheral bloodsamples were collected in 4 EDTA-coated tubes to a totalvolume of 12 mL. Samples were centrifuged at 4�C for 8minutes at 1,500 rpm. Plasma was collected and stored at�80�C. The remaining fraction was lysed using 50 mL of redcell lysis buffer for 20 minutes at room temperature. Theresulting mononuclear cell fraction was washed in PBS EDTA2 mmol/L þ 0.5% bovine serum albumin (BSA) and used forfurther analysis. Staging of patients with colorectal cancer wasdone according to the American Joint Committee on Cancer(AJCC) Staging System.

Mouse strains, bone marrow transplants, ectopic, andorthotopic colon carcinoma model

Animal experiments were carried out with the approval ofthe Animal Care Committee and Review Board at the InstitutoGulbenkian de Ciencia (Oeiras, Portugal). In vivo experimentswere carried out on 4- to 8-week-old female nude mice (C57/BL6 background). For bone marrow transplants, nude micereceived a whole body lethal irradiation (800–950 rads) and 24hours later received an intravenous injection of 2–3� 106 bonemarrow mononuclear cells collected from Actin-GFP malemice (C57/BL6 background). Mice were allowed to recoverfor 2 to 4 weeks. After this period peripheral blood sampleswere collected from the facial vein in EDTA-coated tubes

(Multivette 600, Sarstedt) and analyzed by flow cytometry forGFPþ cells. Mice were considered suitable for further experi-ments when the percentage of GFPþ cells in the peripheralblood was more than 80% of total cells. Xenografted ectopiccolon carcinoma tumors were induced by inoculation of5 � 106 HCT15, HCT116, DLD-1, or HT-29 cells (humancolorectal carcinoma cell lines; these were obtained fromAmerican Type Culture Collection, in 2012, and were notpassaged for more than 6 months in our Laboratory) subcu-taneously in nude mice. Tumors were allowed to grow and atspecific time points (1–3 weeks) mice were sacrificed andtumors were collected. Tumors were fixed (10% formalin orparaformaldehyde) and included (paraffin or gelatin, respec-tively), frozen at �80�C for further RNA isolation, ordigested for fluorescence-activated cell sorting (FACS) anal-ysis. Xenografted orthotopic colorectal carcinoma tumorswere induced by inoculation of 1 � 106 HCT15 or HCT116cells, into the visceral cecal wall of nude mice. Peripheralblood samples were collected from the facial vein at differ-ent time points and further processed for flow cytometricanalysis. CD11b-neutralizing antibody in vivo administra-tion was conducted as follows: briefly, 500 mg anti-CD11b(clone 5C6) neutralizing monoclonal antibodies againstCD11b were administered intraperitoneally every 3 daysinto tumor-bearing mice, starting on day 5 postinoculation.

Isolation of bonemarrow–derived cells from the tumorsTumor samples were mechanically fragmented into 2 � 2

mm2 pieces and then digested with collagenase (Sigma-Aldrich, 2 mg/mL in serum-free Dulbecco's Modified EagleMedium (DMEM) for 1 hour at 37�C and 5% CO2. Afterdigestion, tumor cell suspensions were passed through a meshand washed in sterile PBS. Further isolation of tumor cellpopulation was conducted by cell sorting using FACSaria (BDBiosciences). Isolation of tumor-derived GFPþ population wasdone without the use of any antibody staining, whereas iso-lation of Jag2þ and CD11bþ population required previousstaining with fluorochrome-conjugated antibodies (PE anti-mouse Jag2, 131007, BioLegend and FITC anti-mouse CD11b,101205 BioLegend). Antibodies used were diluted 1:100 in PBSþ BSA 0.5% and incubated in the dark with rotation at 4�C for45 minutes.

In vitro co-culture assaysHCT15 cells were cultured at 1 � 104/cm2 cell density in

DMEM-supplementedmedium (GIBCO)with 10%FBS (Sigma-Aldrich). After 24 hours, medium was changed to DMEM-supplemented medium with 2% FBS and 1 � 105 tumor-associated bone marrow–derived cells were added (GFPþ,GFPþJag2þ, GFPþJag2�, or Jag2þ CD11bþ cells). Coculturewas maintained for 48 hours. After this period, tumor-associ-ated bone marrow–derived cells were gently washed from theculture andHCT15 cells collected formRNA extraction or fixedwith 2% paraformaldehyde for 15 minutes for further immu-nocytochemistry staining. g-Secretase inhibitor (GSI; DAPT,Sigma-Aldrich) was added to cocultures at a final concentra-tion of 10 mmol/L and respective controls received DMSO(Sigma-Aldrich).

Caiado et al.

Cancer Res; 73(14) July 15, 2013 Cancer Research4234




Flow cytometryPeripheral blood mononuclear cell (PBMC) fractions

derived from patients with colorectal cancer were stained forCD11b and Jag2. Briefly, 5 � 105 cells were blocked for 10minutes at 4�C with FcR fragment in a 1:100 dilution and thenincubated with anti-CD11b (APC anti-human CD11b, 301409BioLegend) and Jag2 (PE anti-human Jag2, 346903 BioLegend)antibodies in a 2.5:100 dilution in PBS þ BSA 0.5% for 45minutes in the dark with rotation at 4�C. Characterization oftumor-associated bone marrow–derived cells was conductedby staining digested tumor samples for Jag2 (PE anti-mouseJag2, 131007, BioLegend), CD11b (FITC anti-mouse CD11b,101205 BioLegend), Gr-1 [APC/Cy7 anti-mouse Ly-6G/Ly-6C(Gr-1), 108423 BioLegend], F4/80 (FITC anti-mouse F4/80,123107 BioLegend), Sca-1 (FITC anti-Mouse Ly-6A/E, 557405BD Pharmingen), c-Kit [APC/Cy7 anti-mouse CD117 (c-kit),105825, BioLegend], CD3 (FITC anti-mouse CD3, 100203 Bio-Legend), and CD19 (Alexa Fluor 488 anti-mouse CD19, 115524BioLegend). Antibodies used were diluted 1:100 in PBS þ BSA0.5% and incubated in the dark with rotation at 4�C for 45minutes. Flow cytometry was conducted on FACSCalibur andanalyzed with FlowJo 8.7 Software (Tree Star, Inc. 1997–2012).

Quantitative reverse-transcription PCRRNA extraction (TRIzol, Invitrogen) and cDNA synthesis

[Reverse-transcription with Superscript II reverse transcrip-tase (Invitrogen)] were conducted following standard proto-cols. Reverse-transcription PCR (RT-PCR) was conducted withPower SYBR Green PCRMaster Mix in 7900HT Fast Real-TimePCRSystem (both fromAppliedBiosystems). Primer sequencesfor hE-Cadherin, hVimentin, hHey1, hHey2, mDll1, mDll4,mJag1, and mJag2 are shown in Table 2. The housekeeper geneused to normalize human samples was h18s and to normalizemouse samples was m18s. RT-PCR data were analyzed byDataAssist software (Applied Biosystems).

Tumor histocytochemistry procedure and analysisHuman colorectal cancer tumor paraffin-included samples

were provided by the Pathology department at IPO-Lisboa.Samples were serially sectioned (3 mm), adsorbed into slidesand then subjected to antigen retrieval (PT Link, Dako). Afterbeing deparaffinized, sections were blocked in PBSþ 10% goatserum for 30minutes at room temperature and then incubatedwith primary antibody at room temperature for 1 hour. Coun-terstaining was conducted using Mayer hematoxylin. Serialsections were stained for Jag2 (sc-56041 Santa Cruz, 1:15),CD11b (HPA002274 Sigma, 1:300), E-cadherin (18-0223 Invi-trogen, 1:50), and cytokeratin-19 (CK19; M0888 Dako, 1:50).Slides were analyzed and photographed in a Leica DMD108microscope. Concerning CD11bþ Jag2þ cell quantification,tissue sections were screened at low power field (�100 mag-nification), and the 10 areas with the most intense staining forCD11b (hot spots) were selected. Jag2 staining in these areaswas confirmed in the serial slide. Counts of the hot spots wereconducted at high power field (HPF;�400). The mean numberof positive cells in the 10 hot spot areas was expressed for eachcondition. E-cadherin quantification on each slide was con-ducted using the ImmunoMembrane software version 1.0i (16).

Results were further validated by a pathologist at IPO-Lisboa.Mouse tumors samples were included in either gelatin orparaffin. Paraffin tumor sections were further subjected toantigen retrieval protocols (PT high). Tumor cryosections wereblocked with a 5% FBS/0.1% BSA solution in PBS for 30minutes. Slides were then covered with primary antibodies:anti-mouse PECAM (553369, Pharmingen), anti-mouse CD11b(550282, Pharmingen), anti-human E-cadherin (M3612, Dako);anti-human CK19 (M0888, Dako), anti-human (M0851, Dako),anti-human C-Kit (A4502, Dako), anti-mouse B220 (553085,Pharmingen), and anti-human Jag2 (AbCam). After overnightincubation at 4�C, tumor sections were washed in PBS andincubated with secondary antibodies from Invitrogen (anti-rat-FITC, anti-mouse-Alexa568, anti-rabbit-Alexa488, respec-tively) for 2 hours at room temperature. For the quantificationof GFPþ lineageþ cells in tumor samples, the total number ofGFPþ lineageþ cells was quantified in 5 HPFs (�400) and thendivided by total GFPþ cells. Quantification of vimentinþ cellswas preformed by direct count of vimentin-expressing cellsin 5 HPFs (�400). Quantification of E-cadherin expression ontumor samples was performed by direct quantification ofstaining intensity and area using ImageJ software.

Statistical analysisResults are expressed as mean � SEM. Data were analyzed

with GraphPad software (GraphPad Software, Inc; v4.0b) usingunpaired two-tailed student t test orMann–Whitney test whenindicated. P values of less than 0.05 were considered statisti-cally significant.

ResultsBone marrow–derived cells infiltrate ectopic colorectaltumors and localize in regions undergoing EMT

To characterize the contribution of bone marrow–derivedcells during colorectal cancer progression, we started byinoculating HCT15 (human colon carcinoma cell line) subcu-taneously into nude mice that had been transplanted withactin-GFPþ BM (Supplementary Fig. S1). Tumors were allowedto grow for 1 to 3 weeks (termed early and late tumors,respectively), after which the mice were sacrificed and tumorscollected. As shown in Fig. 1, bonemarrow–derived cells (GFPþ

cells) actively infiltrate early and late tumors, and are found athigher frequency in late tumors (Fig. 1A).We also observed thatas tumors grew, there was a significant decrease in E-cadherinexpression and a significant increase in the expression of themesenchymal marker vimentin (Fig. 1B and C), suggestive ofEMT. Considering that bonemarrow–derived cells' infiltrationinto tumors and EMT were associated with tumor growth, weanalyzed possible correlations between GFPþ cell infiltrationand E-cadherin and vimentin expression in tumor tissues.There was an inverse correlation (P ¼ 0.0097; R2 ¼ 0.7678, 7samples) between GFPþ and E-cadherinþ areas, but a directcorrelation (P¼ 0.0297; R2¼ 0.6448, 7 samples) between GFPþ

areas and the number of vimentinþ cells in tumor tissues (Fig.1D). Detailed observation of tumor sections showed that CK19-positive tumor cells in close proximity with GFPþ cells acquirethe expression of vimentin, while tumor cells further away donot (Fig. 1E, bottom, white arrows). Consistent with the EMT

Bone Marrow Cells Induce EMT

www.aacrjournals.org Cancer Res; 73(14) July 15, 2013 4235




phenotype, tumor cells in proximity of GFPþ cells also showdecreased E-cadherin expression (Fig. 1E bottom right, whitearrow). These data suggest that colorectal cancer growth isassociated with an increased frequency of bone marrow–derived cells infiltration, which in turn correlate with the onsetof EMT. Considering this, we hypothesized bone marrow–derived cells could induce EMT on epithelial tumor cells.

Bone marrow–derived cells induce EMT on colorectalcarcinoma cells in a Notch pathway-dependent manner

To test the ability of bone marrow–derived cells to induceEMT on tumor cells, we conducted in vitro coculture experi-ments with HCT15 cells and bone marrow–derived (GFPþ)cells isolated (sorted out) from late subcutaneous tumors. Asdepicted in Fig. 2A in the presence of bone marrow–derived(GFPþ) cells isolated from tumors (TGFPþ cells), HCT15 lose

E-cadherin expression and gain vimentin expression, as mea-sured by quantitative RT-PCR (qRT-PCR). This can also beobserved in the quantification of E-cadherin–expressingHCT15 cells by immunostaining after coculture (Fig. 2B),suggesting that TGFPþ cells induce vimentin expression anddecrease E-cadherin expression at both transcriptional andtranslational level. Next, we looked into the molecular path-ways regulating EMT induced by TGFPþ cells. The TGF-b andthe Notch signaling pathways (19) have been implicated inEMT, thuswe testedwhether these pathways could be involvedin bone marrow–derived cell-induced EMT on colon carcino-ma cells. In agreement, we conducted coculture assays byadding a TGF-b blocking antibody and a Notch pathwayinhibitor (GSI) and determined its effect on HCT15 cells E-cadherin and vimentin expression. TGF-b inhibition had noeffect (data not shown), whereas addition of GSI inhibited the

Early tumorA E

B

C

D

GFP

GFP E-Cadherin Merge

GFP CK19 Merge



GFP Vimentin Merge

GFP Vimentin Merge

GFP Vimentin Merge

GFP

GFPCK19 GFPVIM GFPE-CAD

Late tumor

Early tumor Late tumor



P = 0.0297r 2 : 0.6448

P = 0.0097r 2 : 0.7678

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0

*

*

*

17.5

15.0

12.5

10.0

7.5

5.0

2.5

0.0

35

30

25

20

15

10

5

0

35

30

25

20

15

10

5

0

40

30

20

10

0

50

40

30

20

10

0

−10

% G

FP

+ a

rea

% E

CA

D+

are

a

EC

AD

are

a (

%)

GFP area (%)

2.5 5.0 7.5 10.0 12.515.0 17.5 20.0

GFP area (%)

Num

ber

of V

im+

ce

lls/x

20

0 H

PF

Num

ber

of V

im+

cells

/x200 H

PF

Earl

y t

um

or

Late

tu

mo

rE

arl

y t

um

or

Late

tu

mo

r

Figure 1. Bone marrow–derived cells infiltrate ectopic colorectal tumors and localize in regions undergoing EMT. A, representative image of early (1 week)and late (3–4 weeks) subcutaneous colon tumors implanted into GFPþ BM nude mice. Scale bar, 100 mm. Quantification of the GFP area percentage in bothearly and late tumors using ImageJ software. B, representative image of GFP (green) and E-cadherin (red) expression on early and late colon tumors.Dashed line delimitates tumor regions that show decreased E-cadherin (ECAD) expression. Scale bar, 100 mm. Quantification of the E-cadherin areapercentage in both early and late tumors using ImageJ software. C, representative image of GFP (green) and vimentin (red) expression on early and latetumors. Scale bar, 100 mm. Quantification of the number of vimentin þ cells in both early and late tumors using ImageJ software. D, correlation plotbetween E-cadherin area percentage and GFP area percentage (on the left) and also between the number of vimentin þ cells and GFP area percentage(on the right). E, representative image of sequential tumor sections of the same region stained for GFP andCK19, GFP and vimentin, andGFP and E-cadherin.Arrows point to tumor cells (CK19þ) in close contact with GFP cells that acquire vimentin expression and lose E-cadherin expression. Scale bar, 10 mm.Data are means � SEM, �, P < 0.05; n ¼ 7 (3 mice in early tumor group and 4 in late tumor group).

Caiado et al.





TGFPþ cell-induced decrease in E-cadherin and increase invimentin expression (Fig. 2A). Furthermore, confirming theinvolvement of the Notch pathway is the observation thatHCT15 cells express Notch receptors 1 and 4 (data not shown)andmore importantly the Notch pathway downstream targetsHey 1 and 2 are upregulated inHCT15 cells undergoing EMT, inthe presence of TGFPþ cells (Fig. 2A). These data suggest thatbone marrow–derived cells induce EMT via Notch pathwayactivation on tumor cells.

Bone marrow–derived cells expressing Jag2 induceEMT on colorectal cancer cellsHaving shown TGFPþ cells induce EMT on HCT15 cells

and that this process involved Notch pathway activation,next we determined which ligands of the Delta:Notch family

were expressed by the tumor-infiltrating bone marrow–derived cells. We determined and compared the expressionof the ligands Delta-like ligand 1 (Dll1), 4 (Dll4), and Jag1 and2 (Jag1, Jag2) in bone marrow–derived cells (GFPþ) collectedfrom late tumors and from the corresponding bone marrowsamples. Jag2 was the most abundantly expressed ligand onTGFPþ cells, its expression being significantly higher onTGFPþ cells than on those isolated from bone marrow (Fig.3A). Detailed flow cytometry analysis of tumor and bonemarrow samples of tumor-bearing mice revealed a popula-tion of GFPþ Jag2þ cells in both tissues, although at a higherfrequency in the tumors, representing an average of 5.5 �1.12% cells (Fig. 3B). Taking into account the high frequencyof GFPþ Jagþ cells in tumors, next we investigated whetherthis population was able to induce EMT on HCT15 colon

3

2

1

0

1,2001,1001,000

900800700600500400300200100

0

HCT15

C

HCT15

+GSI

HCT15

+TGFP+

HCT15

+GSI+

TGFP+

HCT15

C

HCT15

+GSI+

HCT15

+TGFP+

HCT15

+GSI+

TGFP+

HCT15

C

HCT15

+GSI

HCT15

+TGFP

HCT15

+TGFP+G

SI

HCT15

C

HCT15

+GSI+

HCT15

+TGFP+

HCT15

+GSI+

TGFP+

HCT15

C

HCT15

+GSI

HCT15

+TGFP+

HCT15

+GSI+

TGFP+

Eca

d G

en

e e

xp

ressio

n

(ra

tio

to

18

s)

Vim

Ge

ne

exp

ressio

n

(ra

tio

to

18

s)

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

HCT15 C

A

B

E-Cadherin E-Cadherin

E-Cadherin

GFP GFP

E-Cadherin

HCT15 C + TGFP HCT15 C + TGFP + GSI

HCT15 C + GSI

Hey1

Ge

ne

exp

ressio

n

(ra

tio

to

18

s)

% E

CA

D+

HC

T15 c

ells

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

60

50

40

30

20

10

0

Hey2

Ge

ne

exp

ressio

n

(ra

tio

to

18

s)

Figure 2. Bone marrow–derivedcells induce EMT on coloncarcinoma cells in a Notchpathway-dependent manner.A, quantification of E-cadherin,vimentin, Hey 1, and Hey 2 geneexpression (normalized to 18srRNA expression) by qRT-PCR onHCT15 alone (HCT15 C), in thepresence of 10 mmol/LGSI (HCT15þ GSI), in the presence of bonemarrow–derived cells isolatedfrom the tumors (HCT15 þ TGFP)alone, or in the presence of 10mmol/L GSI (HCT15 þ TGFP þGSI). B, quantification of E-cadherinþ HCT15 cells on HCT15C, HCT15 þ GSI, HCT15 þ TGFP,and on HCT15 þ TGFP þ GSIcoculture conditions.Representative images ofE-cadherin (red) expression inHCT15 cells cocultured with bonemarrow–derived cells isolatedfrom the tumors (GFPþ; green).Data shown are means � SEM of3 independent experiments (n¼ 3),�, P < 0.05. Scale bar, 50 mm.






carcinoma cells. GFPþ Jag2þ, and also GFPþ Jag2� cells weresorted from late tumors (Fig. 3B) and cocultured withHCT15 in the presence or absence of GSI. As depictedin Fig. 3C and D, GFPþ Jag2þ but not GFPþ Jag2� cellswere able to reduce E-cadherin transcription in a Notchpathway-dependent manner. In accordance, the number ofHCT15 E-cadherinþ cells is significantly reduced whenthese are cocultured in vitro with tumor GFPþJag2þ (Fig.3C). This phenotype is reversed upon the addition of GSI orwhen HCT15 cells are cocultured with GFPþ Jag2 negativecells (Fig. 3E). Taking together these results suggest thatbone marrow–derived cells modulate EMT via Jag2 medi-ated Notch-pathway activation on tumor cells.

Bone marrow–derived CD11bþJag2þ cells induce EMTvia Jag2-mediated Notch pathway activation

Having shown that bone marrow–derived Jag2þ cells areresponsible for tumor cell EMT via Notch pathway activation,next we sought to determine the identity of these cells moreprecisely. The majority of TGFPþ Jag2þ cells were CD11bþ (85� 1.0%), F4/80þ (64.1� 0,55%), and Sca-1þ (52.5� 1.55%) (Fig.4A, B, and C). Considering the high frequency of CD11bþ cellswithin the TGFPþ Jag2þ population,we testedwhether tumor--derived CD11bþ Jag2þ cells (CD11bþJag2þ) could induceHCT15 EMT in vitro. Accordingly, coculture of HCT15 tumorcells with tumor-derived CD11bþJag2þ cells led to a significantdecrease in E-cadherin expression at both the mRNA and

11

10

9

8

7

6

5

4

3

2

1

0

BM T

HCT15

C

Jag1

BM

Jag1

T

Jag2

BM

Jag2

T

DII1

BM

DII1

T

DII4

BM

DII4

T

HCT15

+GSI

HCT15

+TGFP+J

AG2+

HCT15

+GSI+

TGFP+J

AG2+

HCT15

+TGFP+J

AG–

HCT15

C

HCT15

+GSI

HCT15

+TGFP+J

AG2+

HCT15

+GSI+

TGFP+J

AG2+

HCT15

+TGFP+J

AG2–

HCT15

C

HCT15

+GSI

HCT15

+TGFP+J

AG2+

HCT15

+GSI+

TGFP+J

AG2+

HCT15

+TGFP+J

AG2–

Eca

d G

en

e e

xp

ressio

n

(ra

tio

to

18

s)

Ge

ne

exp

ressio

n

(ra

tio

to

18

s)

GF

P

Jag2 PE

HCT15 C

A B

C

E

D



GFP

E-Cadherin

GFP

GFP

HCT15 + TGFP + JAG2+

HCT15 + TGFP + JAG2–

HCT15 + TGFP + JAG2+ + GSI

HCT15 + GSI

Hey1

Ge

ne

exp

ressio

n

(ra

tio

to

18

s)

% E

CA

D+

HC

T15 c

ells

% G

FP

+ J

AG

2+ c

ells

6.56.05.55.04.54.03.53.02.52.01.51.00.50.0

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0.00

55

50

45

40

35

30

25

20

15

10

5

0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

Tumor:104

103

102

101

100

100 101 102 103 104

Figure 3. Bone marrow–derivedcells expressing Jag2 induce EMTon colorectal cancer cells. A, Jag1,Jag2, Dll1, and Dll4 geneexpression (normalized to 18srRNA expression), determined byqRT-PCR in GFPþ cells collectedfrom tumors or bone marrowsamples. Data shown are means�SEMof 3 independent experiments(n ¼ 3); �, P < 0.05 (betweenindicated groups); #, P < 0.05(between gene expression in bonemarrow or tumor samples). B,quantification of the percentage ofGFPþ Jag2þ cells in bonemarrow and tumor samples.Representative flow cytometry plotshowingJag2þbonemarrow (BM)–derived cells in tumor (T) sample.C and D, quantification ofE-cadherin (C) and Hey 1 geneexpression (normalized to 18srRNA expression; D) by qRT-PCRon HCT15 alone (HCT15 C), in thepresence of 10 mmol/L GSI (HCT15þ GSI), in the presence of tumorassociated bone marrow–derivedJag2þ cells (HCT15 þ TGFP-Jag2þ), in the presence of 10 mmol/L GSI and tumor-associated bonemarrow–derived Jag2þ cells(HCT15 þ TGFP þ Jag2þ þ GSI),or in the presence of tumorassociated bone marrow–derivedJag2–negative cells (HCT15 þTGFP-Jag2�). E, quantification ofE-cadherinþ cells on HCT15C,HCT15þGSI,HCT15þTGFPþJag2þ, HCT15 þ TGFP-Jag2þ þGSI, or HCT15 þ TGFP-Jag2-(negative) conditions. Panel showsrepresentative images ofE-cadherin (red) expression inHCT15cells aloneor in the differentcoculture conditions. Bonemarrow–derived Jag2þ cellsisolated from the tumors are GFPþ

(green). Data shown are means �SEMof 3 independent experiments(n¼3), �,P<0.05.Scalebar,50mm.

Caiado et al.





protein level (Fig. 4D and E), in a Notch pathway-dependentmanner, as GSI treatment reverted this effect. Altogether thesedata suggest that CD11bþJag2þ bone marrow–derived cellsactively infiltrate colorectal cancer tumors and induce EMT ontumor cells in a Notch pathway-dependent manner.

CD11bþJag2þ cells are mobilized to PB and home totumor tissues in different colorectal cancer modelsConsidering the observed effect of bone marrow–derived

CD11bþJag2þ cell population in inducing EMT in HCT15cells in vitro and in vivo, next we quantified the recruitmentof this population in other colon cancer models. For thispurpose, we developed subcutaneous colon carcinoma xeno-

transplants using 3 human colorectal cancer cell lines,HCT15, DLD-1, and HT-29, and evaluated the presence ofCD11bþJag2þ cells in the peripheral blood and in the tumortissues. There was a significant increase in the frequencyof CD11bþJag2þ cells in the peripheral blood comparedwith control mice (no tumor) in all models (Fig. 5A), thisbeing particularly evident on HCT15-xenotransplanted mice,which showed a significant increase on days 7 and 14 aftertumor inoculation. Moreover, FACS analysis of tumor sam-ples showed that CD11bþJag2þ cells are present in tumorxenotransplants derived from all cell lines at a frequencyranging from 4.5%–8.5% of total cells (Fig. 5B andC). To furthervalidate the biologic significance of CD11bþJag2þ cells in

HCT15 C

T CD11b

CD11b

CD11b G

r-1

F4/80

Sca-1

T F4/8

0

T Sca

-1

T C-K

it

C-Kit

T CD3

CD3

T CD19

CD19

BM C

D19

BM C

D3

BM C

-Kit

BM S

ca-1

T CD11bG

r1

BM C

D11b

BM C

D11bGr1

BM F

4/80

HCT15+GSI

HCT15+CD11b+ JA

G2+

HCT15+GSI+

CD11b+ JA

G2+

HCT15 C

HCT15+GSI

HCT15+CD11b+ JA

G2+

HCT15+GSI+

CD11b+ JA

G2+

Gene e

xp

ressio

n

(ratio

to

18

s)

CD

11b F

ITC

Jag2 PE

Ecad

Hey1

HCT15 C

A

B

E

D

C


E-Cadherin

CD11b

E-Cadherin

CD11b

HCT15 + CD11b+JAG2+ HCT15 + CD11b+JAG2+ GSI

HCT15 + GSI

% E

CA

D+

HC

T15 c

ells

% J

AG

2+ L

ine

ag

e+

ce

lls%

Lin

ea

ge

+ fra

ctio

n

in t

ota

l tu

mo

r JA

G2

+ c

ells

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

90

80

70

60

50

40

30

20

10

0

60

50

40

30

20

10

0

2.0

1.5

1.0

0.5

0.0

Tumor:

104

105

103

102

0

0 102 103 104 105

Figure 4. Bone marrow–derivedCD11bþJag2þ cells induce EMTvia Jag2–mediatedNotch pathwayactivation. A, quantification ofJag2þ CD11bþ/CD11bþ Gr-1þ/F4/80þ/Sca-1þ/c-Kitþ/CD3þ/CD19þ expressing cells in tumorsand corresponding bone marrowsamples. B, quantification of thepercentage of tumor-infiltratingJag2þ cells that express CD11b/CD11b Gr-1/F4/80/Sca-1/c-Kit/CD3 or CD19 marker. C,representative flow cytometry plotshowing CD11bþJag2þ BM-derived cells. D, quantification ofE-cadherin and Hey 1 geneexpression (normalized to 18srRNA expression) by qRT-PCR onHCT15 alone (HCT15 C), in thepresence of 10uM GSI (HCT15 þGSI), in the presence of bonemarrow–derived CD11bþJag2þ

cells isolated from tumors(HCT15 þ CD11bþJag2þ), and inthe presence of 10 mmol/L GSI andtumor associated bone marrow–

derived CD11bþJag2þ cells(HCT15 þ CD11bþJag2þ þ GSI).E, quantificationof E-cadherinþonHCT15 C, HCT15þGSI, HCT15þCD11bþJag2þ, and HCT15 þCD11bþJag2þ þ GSI groups.Representative images of E-cadherin (red) expression inHCT15 cells and CD11b (green)expression on tumor associatedCD11bþ Jag2þ cells. Panelshows representative pictures ofcoculture experiments whereE-cadherin is labeled in red andCD11b bonemarrow–derived cellsare labeled in green. Scale bar,50 mm. Data shown aremeans � SEM of 3 independentexperiments (n ¼ 3), �, P < 0.05.






CD

11

b F

ITC

JAG2 PE

Control HCT15

Control HCT15 HCT116

W2

W4

W6

W8

W10

HCT15 W10 HCT116 W10

HCT15 HCT116

HCT15 D14

Ec

top

ic t

um

or

Ort

ho

top

ic t

um

or

DLD-1

DLD-1 D14

HT-29 HCT15 DLD-1 HT-29

HT-29 D14

Day7

Day14

A B

C

ED

F

5.55.04.54.03.53.02.52.01.51.00.50.0

2.0

1.5

1.0

0.5

0.0

9

5

7

6

5

4

3

2

1

0

104

105

103

102

0

CD

11

b F

ITC 104

105

103

102

0

CD

11

b F

ITC 104

105

103

102

0

0 102 103 104 105

CD

11

b F

ITC

JAG2 PE

104

103

102

101

100

CD

11

b F

ITC

104

103

102

101

100

102101 103 104100

JAG2 PE102101 103 104100

JAG2 PE

0 102 103 104 105

JAG2 PE0 102 103 104 105

% C

D1

1b

+Ja

g2

+ c

ells

in

PB

MN

Cs

6.56.05.55.04.54.03.53.02.52.01.51.00.50.0%

CD

11b

+Ja

g2

+ c

ells

in

tu

mo

rs

% C

D11b

+Ja

g2

+ c

ells

in

PB

MN

Cs

% C

D1

1b

+Ja

g2

+ c

ells

in

tu

mo

rs

Figure 5. CD11bþJag2þ cells are mobilized to PB and home to tumor tissues in different colorectal cancer models. A, flow cytometry–based quantification ofCD11bþJag2þ percentage in PBMC fraction of control (no tumor) and tumor-bearing mice xenotransplanted with HCT15, DLD-1, and HT-29 colorectal celllines, 7 and 14 days after ectopic tumor inoculation. B, quantification of CD11bþJag2þ percentage in ectopic HCT15, DLD-1, and HT-29 tumor samples.C, representative flow cytometry plot showing CD11bþJag2þ cells in ectopic HCT15, DLD-1, and HT-29 tumor samples. D, quantification of CD11bþJag2þ

percentage in peripheral blood mononuclear cells' (PBMC) fraction of control and tumor-bearing mice xenotransplanted with HCT15 and HCT116colorectal cell lines at week 2 to 10 after orthotopic tumor inoculation. E, quantification of CD11bþJag2þ percentage in orthotopic HCT15 and HCT116 tumorsamples. F, representative flow cytometry plot showing CD11bþJag2þ cells in orthotopic HCT15 and HCT116 tumor samples. Data are means � SEM;�, P < 0.05; n ¼ 12 (3 mice per group in ectopic models) and n ¼ 12 (4 mice per group in orthotopic models).

Caiado et al.





colorectal cancer growth, we developed orthotopic models ofcolorectal cancer using HCT15 and HCT116 cell lines andevaluated their presence in the peripheral blood and in tumortissues, as above. Mice bearing orthotopic HCT15 and HCT116tumors showed increased frequency of peripheral bloodCD11bþJag2þ cells compared with control mice on weeks 4and 6, after tumor inoculation (Fig. 5D). Moreover, FACSanalysis of tumor samples shows that CD11bþJag2þ cells arepresent in orthotopic tumors at a frequency ranging from 0.5%to 6% of total tumor cells (Fig. 5E and F). Together, these datashow that in different murine models of colorectal cancer,CD11bþJag2þ cells are mobilized to the peripheral blood andare actively recruited into tumors, which is suggestive of thebiologic significance of this population in colorectal cancergrowth and EMT onset.

CD11b-neutralizing antibodies reduce CD11bþJag2þ

cell recruitment, resulting in decreased tumor growthand EMT in vivoTo further test the role of CD11bþJag2þ in colorectal cancer

progression and EMT, we treated mice bearing ectopic HCT15tumors with neutralizing monoclonal antibodies againstCD11b. CD11b neutralization caused a significant reductionin tumor growth (Fig. 6A) and also on the peripheral bloodlevels of CD11bþ and CD11bþJag2þ cells compared with micetreated with vehicle alone (Fig. 6B). Moreover, flow cytometryand immunostaining-based quantification of CD11bþ andCD11bþJag2þ cells in tumor tissues showed that CD11b neu-tralization led to a significant reduction in both cell popula-tions (Fig. 6C–E). Having shown that CD11b neutralizationleads to a significant reduction in the number of peripheralblood and tumor-infiltrating CD11bþJag2þ cells, we deter-mined its effect in the onset of EMT. As shown in Fig 6, whilecontrol (untreated) mice show the expected decrease in E-cad-herin expression (Fig. 6F and G), this is sustained and evenslightly increases in tumors of mice treated with CD11b-neutralizing antibody. Analysis of Notch pathway downstreamtargets Hey 1 and Hes 1 expression showed the reduced EMTonset in tumors treated with anti-CD11b–neutralizing anti-body is accompanied by decreased activation of the Notchpathway (Fig. 6F), which is in accordance with our in vitro data.Taken together, these data suggest that treatment of ectopiccolon cancer-bearing mice with neutralizing monoclonalantibodies against CD11b reduces infiltration of CD11bþJag2þ

cells into the tumors, reducing EMT and reducing Notchpathway activation.

CD11bþJag2þ PB and tumor levels correlate with lowerE-cadherin expression andmetastatic disease in patientswith colorectal cancerHaving shown the importance of bone marrow–derived

CD11bþJag2þ cell population in inducing EMT in murinecolorectal cancer models, and the easy quantification of thesecells in peripheral blood of colon cancer-bearing mice, nextwe tested the feasibility and usefulness of quantifying thesecells in the samples of patient with colorectal cancer. Wequantified the levels of CD11bþJag2þ cells in peripheral bloodsamples of 40 patients with colorectal cancer diagnosed at

different stages (according to the AJCC Staging System). Forthe analysis, patients were classified as: TxN0M0 (patients withcolorectal cancer without lymph node or distant metastasis,stages I–II of the AJCC), TxNxM0 (patients with colorectalcancer with lymph node metastasis and without distant meta-stasis, stage III of the AJCC), and TxNxMx (patients withcolorectal cancer with distant metastasis, stage IV of theAJCC). We observed a significant correlation between PBCD11bþJag2þ cell levels and colorectal cancer stages, withhigher stage colorectal cancer patients showing higher peri-pheral blood CD11bþJag2þ levels (Fig. 7A and B). Moreover,there was an inverse correlation (P ¼ 0.0197; R2 ¼ 0.4708, 11patients) betweenperipheral bloodCD11bþJag2þ cell levels andtumor E-cadherin expression (Fig. 7C). We were also able toinvestigate the relation betweenCD11bþJag2þ tumor levels andcolorectal cancer staging in 12 patients. We observed thatTxNxM0 and TxNxMx stage patients had significantly highernumbers of CD11bþJag2þ cells in tumor samples relativelyto TxN0M0 patients (Fig. 7D and E). We also observed astrong inverse correlation (P¼ 0.0007; R2¼ 0.7011, 12 patients)between CD11bþJag2þ numbers in tumors and tumor E-cad-herin expression levels (Fig. 7F). Importantly, in our patientcohort, there was no correlation between tumor size and thepresence of peripheral blood or tumor CD11bþJag2þ cells(Supplementary Fig. S2). Together, these data strongly suggestthat quantification of both CD11bþJag2þ cells in the PB andin the tumor samples of patients with colorectal cancer corre-lates with colorectal cancer staging, and thus may be used as aprognostic or diagnostic marker. These data also validate ourdata obtained from colorectal cancer mouse models, where weshowed CD11bþJag2þ cells infiltrate colorectal cancer tumorsand induce EMT.

DiscussionOver the last decade, there has been an increase in our

understanding of the contribution of tumor stromal compo-nents in the regulation of tumor progression and metastasisformation (17). EMT has been shown to be essential forcarcinoma progression, namely in breast, colon, and prostatecarcinoma, preceding invasion and metastasis formation(3, 18). One of the major molecular regulators of EMT isE-cadherin. E-cadherin is a member of the cadherin family ofhomophilic cell adhesion molecules and is essential for themaintenance of adherens junctions that confer physical integ-rity and polarization to epithelial cells. Targeted disruption ofE-cadherin during tumor progression resulting in decreasedintercellular adhesiveness is one of the most common altera-tions in human cancers (19, 20). In fact, E-cadherin functionalinactivation represents a critical step in the acquisition ofinvasive capacity by epithelial tumor cells. Accordingly, abo-lishing E-cadherin function in vitro confers invasive propertiesto noninvasive cells and conversely, introduction of E-cadherininto invasive epithelial cell lines abrogates their invasivepotential. Not surprisingly, then, loss of E-cadherin expressionis a defining feature of EMT (9). Although many molecularregulators of EMT have been identified, the cellular interac-tions between tumor cells and tumor stromal cells responsiblefor tumor EMT are still unclear.






Ecad G

ene e

xpre

ssio

n

(ratio t

o 1

8s)

% E

CA

D+

Are

aA

C

D

E

F

G

B

E-Cadherin

Jag2

E-Cadherin

CD11b

Jag2

CD11b

CD11b CD11b

Hey1 G

ene e

xpre

ssio

n

(ratio t

o 1

8s)

Hes1 G

ene e

xpre

ssio

n

(ratio t

o 1

8s)

Tu

mo

r vo

lum

e (

mm

3)

% C

ell

popula

tion in P

BM

Cs

% C

ell

popula

tion in t

um

ors

Num

ber

of C

D11b

+

cells

/ 4

00x H

PF

Num

ber

of C

D11b

+JA

G2

+

cells

/ 4

00x H

PF

1,4001,3001,2001,1001,000

900800700600500400300200100

0

45

40

35

30

25

20

15

10

5

0

17.5

15.0

12.5

10.0

7.5

5.0

2.5

0.0

75

50

25

0

50

40

30

20

10

0

0.45

0.40

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0.00

45

40

35

30

25

20

15

10

5

0

2.25

2.00

1.75

1.50

1.25

1.00

0.75

0.50

0.25

0.00

25

20

15

10

5

0

T D15

*

**

*

*

*

*

*

*

*

T + aCD11b

TControl T + aCD11b

T T + aCD11b

T T + aCD11b

T T + aCD11b

T T + aCD11b

T

T + a

CD11

b T

T + a

CD11

b T

T + a

CD11

b

T + aCD11b D15T D15

T + aCD11bT D15

CD11b+

CD11b+JAG2+

CD11b+

CD11b+JAG2+

T

T a-C

D11

b

Figure 6. CD11b-neutralizingantibodies in vivo administrationreduces CD11bþJag2þ cellrecruitment, resulting in decreasedtumor growth and EMT. A, ectopictumor volume quantification inPBS (T) or anti-CD11b (T þa-CD11b)–treated mice, 15 daysafter inoculation. Representativeimage of tumors collected at day15 from control and anti-CD11b–treated mice. B, flow cytometry–based quantification of thepercentage of CD11bþ andCD11bþ Jag2þ cells in themononuclear cell fraction ofperipheral blood samples ofcontrol (no tumor), PBS (T), andanti-CD11b (T þ a-CD11b)–treated mice, 15 days afterinoculation. C, flow cytometry–based quantification of thepercentage of CD11bþ andCD11bþ Jag2þ cells in tumorsamples of PBS (T) and anti-CD11b (T þ a-CD11b)–treatedmice, 15 days after inoculation.D, immunohistologicquantification of CD11bþ cells intumor samples of PBS (T) and anti-CD11b (T þ a-CD11b)–treatedmice per �400HPF, 15 days afterinoculation. RepresentativeimagesofCD11bþcells (green) in Tand T þ aCD11b–treated micetumor samples. Scale bar, 50 mm.E, immunohistologic quantificationof CD11bþ Jag2þ cells in tumorsamples of PBS (T) and anti-CD11b (T þ a-CD11b)–treatedmice per �400 HPF, 15 days afterinoculation. Representativeimages of CD11bþ (green) Jag2þ

(red) cells in T and T þ aCD11btreatedmice tumor samples. Scalebar, 50 mm. F, quantification of E-cadherin, Hey 1, and Hes 1 geneexpression (normalized to 18srRNA expression) by qRT-PCR intumor samples of PBS (T) and anti-CD11b (T þ a-CD11b)–treatedmice. G, quantification of theE-cadherin area percentage intumor samples of PBS (T) and anti-CD11b (T þ a-CD11b)–treatedmice. Representative images ofE-cadherinþ cells (red) in T and TþaCD11b–treated mice tumorsamples. Scale bar, 100 mm. Dataare means � SEM; �, P < 0.05;n ¼ 12 (4 mice per group: control,T, and T þ a CD11b).

Caiado et al.





TxN0M0 TxNxM0 TxNxMx




0 10 20 30 40 50 60 70

A

B

C

ED

F

CD

11

b A

PC

104

103

102

101

100

CD

11

b A

PC

104

103

102

101

100

CD

11

b A

PC

104

103

102

101

100

JAG2 PE

102101 103 104100

JAG2 PE

102101 103 104100

JAG2 PE

102101 103 104100

656055504540353025201510

50

155150145140135130125120115110105100959085

20

19

18

17

16

15

20

19

18

17

16

15

14

13

Nu

mb

er

of

CD

11

b+JA

G2

+

ce

lls /

ul P

B

Nu

mb

er

of C

D1

1b

+JA

G2

+

ce

lls /

40

0x H

PF

Number of CD11b+JAG2+ cells / ul PB

80 90 100 110 120 130 140 150 160

Number of CD11b+JAG2+

cells / 400x HPF

P = 0.0197

r 2 = 0.4708

P = 0.0007

r 2 = 0.7011

EC

AD

im

mu

no

sta

inin

g s

co

re

EC

AD

im

mu

no

sta

inin

g s

co

re

EC

AD

JA

G2

CD

11

bC

K1

9

Figure 7. CD11bþJag2þ PB and tumor levels correlate with lower E-cadherin expression and metastatic disease in patients with colorectal cancer. A,quantification of the number of CD11bþJAG2þ cells per mL of peripheral blood samples of patients with colorectal cancer at different stages: TxN0M0(patients without colorectal cancer lymph node or distant metastasis, stages I-II of the AJCC), TxNxM0 (patients with colorectal cancer lymph nodemetastasis and without distant metastasis, stage III of the AJCC), and TxNxMx (patients with colorectal cancer distant metastasis, stage IV of the AJCC).B, representative flow cytometry plot showing CD11bþ Jag2þ cells in PB samples of patients with colorectal cancer at different stages. C, correlationplot between the number of CD11bþJAG2þ cells in circulation and the E-cadherin score determined by immunostaining of primary tumor sections inindividual patients with colorectal cancer. D, immunostaining quantification of CD11bþJAG2þ cells per 400x HPF in primary tumor samples of patients withcolorectal cancer at different stages. E, representative images of CD11b, Jag2, E-cadherin, and CK19 (CK-19) immunostainings in serial sections ofprimary tumor samples of patients with colorectal cancer at different stages. F, correlation plot between the number of tumor CD11bþJAG2þ cell number per�400 HPF and the E-cadherin score determined by immunostaining of primary tumor sections in individual patient with colorectal cancer. Data areindicated as means; �, P < 0.05 using Mann–Whitney test. Scale bar, 50 mm.






Bone marrow–derived cells, have been shown to directlyimpact tumor pathophysiology regulating multiple aspectsof the metastasization process by modulating tumor angio-genesis, promoting tumor cell invasion, extravasation, intra-vasation, and micrometastasis establishment, and growthand vascularization (21). However, a role for bone marrow–derived cells in the first steps of metastases formation,namely in EMT, had not been addressed and was the subjectof this study. In detail, we investigated a putative role forbone marrow–derived cells in regulating EMT in metastaticcolorectal cancer. We show in several ectopic and orthotopiccolorectal cancer models that tumor progression is associ-ated with an increased accumulation of bone marrow–derived cells, mainly of myeloid origin. Furthermore, weobserve that in late tumors there are regions highly infil-trated by bone marrow–derived cells that show evidence forEMT, namely a robust decrease in E-cadherin expression anda significant increase in vimentin expression in CK19þtumor cells. It was highly suggestive from our analysis ofserial tumor slides that tumor cells (CK19þ) in close prox-imity with bone marrow–derived cells (GFPþ) showed thedescribed hallmarks of EMT. This observation led us tospeculate that a direct interaction between bone marrow–derived and tumor cells could modulate EMT in the latter.To test this hypothesis, we used in vitro coculture systems,where colorectal cancer cell lines are cultured with bonemarrow–derived tumor stromal cells (GFPþ). We used thissimple system to investigate the molecular regulation ofEMT induction by bone marrow–derived tumor-associatedcells. Although TGF-b pathway activation has been exten-sively described as an inducer of EMT (22), inhibition ofTGF-b with neutralizing antibodies in our coculture experi-ments failed to inhibit the EMT-inducing capacity of bonemarrow–derived cells. On the other hand, the Notch path-way has also been associated with EMT and metastasisformation in pancreatic (23) and colorectal cancer (24).Moreover, Notch pathway activation was shown in breastcancer brain metastasis (25). Another study shows that inpatients with breast cancer, increased expression of eitherJag1 or Notch1 is predictive of poor overall survival (26).Moreover, in colon cancer, Notch 1 overexpression has beenshown to correlate with pathologic grade, progression, andmetastasis (27). Considering this, we tested the effect of GSI(Notch pathway inhibitor) in reversing the EMT induction bybone marrow–derived cells on colorectal cancer cell lines.GSI addition inhibited EMT induction by bone marrow–derived cells, accompanied by a significant reduction in theexpression of Notch pathway downstream targets Hey 1 and2. Although we did not address Notch downstream effectorsregulating EMT, recent studies suggest the mechanisms bywhich Notch may induce EMT might involve activation of E-cadherin transcriptional repressor Snail2 (28, 29).

Importantly, we show that the bone marrow–derivedtumor-infiltrating cells, which are mainly of myeloid origin(CD11bþ), increased expression of Jag2, but not of otherNotch ligands. Interestingly, Jag2 expression in tumor cellshas been recently described as a major regulator of EMT andmetastases in lung adenocarcinoma via a miR-200–depen-

dent downstream mechanism (30). Furthermore, quantifi-cation of the GFPþ Jag2þ populations present in the bonemarrow and in the tumors revealed a 10-fold increase in thepercentage of this population in tumors, suggesting thatbone marrow–derived Jag2þ cells are actively recruited intotumors. The signal(s) responsible for the selective recruit-ment of this specific bone marrow–derived cell populationinto colon tumors remain undisclosed; we measured themost commonly studied chemokines including SDF1a,MCP1, but these were not actively secreted in our coloncancer models (data not shown). Moreover, recent studiesalso showed that microparticles/microvesicles/exosomesreleased by tumors may communicate and "educate" thebone marrow microenvironment, resulting in selectiverecruitment of bone marrow–derived cells to sites of metas-tases to form the premetastatic niche (31). Whether specificcolon cancer-derived exosomes selectively induce the mobi-lization of CD11bþJag2þ cells is undisclosed. Concerning themolecular mechanisms regulating Jag2 expression, recentdata indicate its expression on breast cancer cells is regu-lated by hypoxia and is directly involved in breast cancermetastases (32). It is therefore legitimate to speculate thatduring colorectal cancer growth, recruited bone marrow–derived CD11bþ cells enter the tumor site and are exposed tosignals from the tumor microenvironment including hypoxiathat regulate the expression of Jag2. As shown here, once theinfiltrating cells express Jag2, these are capable of inducingEMT on subsets of colon cancer cells. The relative contri-bution of Jag2 expressed by infiltrating bone marrow–derived cells and by tumor cells proper for the onset ofEMT is still undisclosed and will be the subject of futurestudies in our laboratory.

These findings led us to hypothesize that selective target-ing of the CD11bþJag2þ bone marrow–derived populationmight delay the onset of EMT, a first step in the metastasiscascade. As we do not presently have access to specificmonoclonal-neutralizing antibodies to Jag2, we sought toimpede the recruitment of CD11bþ cells (which includethe Jag2þ population) into the tumors. Several studiesfollowed a similar approach and concluded that targetingthe CD11bþ population of infiltrating cells may be of ther-apeutic benefit (33, 34). Moreover, Toh and colleagues(35) recently showed myeloid-derived suppressor cells (alsoCD11bþ) promote EMT, although they did not identify themechanism involved. In our colon cancer models, CD11bneutralization led to a significant decrease in CD11bþJag2þ

cell levels in peripheral blood and tumor, delayed EMT,and reduced Notch pathway activation in the tumors.Together, these data strongly suggest that bone marrow–derived CD11bþJag2þ cells represent a targetable cell pop-ulation, which is actively recruited into tumors and inducesEMT in a Notch pathway-dependent manner.

Having shown the biologic significance of CD11bþJag2þ

cells in mouse models of colorectal cancer, we investigatedthe involvement of this cell population in human colorectalcancer progression. We observed that peripheral blood levelsof CD11bþJag2þ cells correlate with colorectal cancer stages,with higher levels of CD11bþJag2þ cells being observed in

Caiado et al.





colorectal cancer patients with metastatic disease (mostlyconsisting of liver metastases), whereas lower CD11bþJag2þ

levels are detected in patients with colorectal cancer with-out metastatic disease. Furthermore, we found a significantcorrelation between CD11bþJag2þ cell peripheral bloodlevels and the levels of E-cadherin expression in the corre-sponding (i.e., patient-specific) tumor tissues. Taking thisinto account, we suggest the CD11bþJag2þ cells are biolog-ically relevant in human colorectal cancer and that mea-suring the peripheral blood levels of CD11bþJag2þ cells inpatients with colorectal cancer may be used as a biomarkerto support colorectal cancer staging.Our data highlight the importance of looking at the initial

steps of the metastasis cascade also in a systemic manner. Indetail, we show a bone marrow–derived CD11bþJag2þ popu-lation can activate Notch signaling on colon cancer cells,resulting in EMT induction in vitro and in vivo. Given therecent excitement from clinical trials using Notch pathwayinhibitors for the treatment of tumors, and particularly aimingat blocking tumor angiogenesis (36, 37), the data presentedhere paves the way for the use of Notch pathway inhibitors toimpede metastases onset, by impeding EMT induction at aprimary tumor site.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were declared.

Authors' ContributionsConception and design: F. Caiado, I. Rosa, H. Yagita, P. Fidalgo, A.D. Pereira,S. DiasDevelopment of methodology: F. Caiado, T. Carvalho, H. YagitaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): F. Caiado, I. Rosa, A. Costa, B. Heissig, K. Hattori, J.P.da Silva, P. FidalgoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): F. Caiado, A. Costa, P. Fidalgo, A.D. Pereira, S�ergioDiasWriting, review, and/or revision of the manuscript: F. Caiado, I. Rosa,H. Yagita, P. Fidalgo, A.D. Pereira, S. DiasAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): F. Caiado, L. Remedio, J. MatosStudy supervision: H. Yagita, A.D. Pereira, S. Dias

AcknowledgmentsThe authors thank the members of the Angiogenesis Lab for their input and

suggestions.

Grant SupportThis study was supported by Fundacao para a Ciencia e Tecnologia (FCT,

Portuguese Government) grants (S. Dias) and fellowships.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 17, 2013; revised April 3, 2013; accepted April 18, 2013;published OnlineFirst May 30, 2013.

References1. Chambers AF, Groom AC, MacDonald IC. Metastasis: dissemination

and growth of cancer cells in metastatic sites. Nat Rev Cancer2002;2:563–72.

2. Fidler IJ. The pathogenesis of cancer metastasis: the "seed and soil"hypothesis revisited. Nat Rev Cancer 2003;3:453–8.

3. HugoH, AcklandML,Blick T, LawrenceMG,Clements JA,WilliamsED,et al. Epithelial–mesenchymal and mesenchymal–epithelial transi-tions in carcinoma progression. J Cell Physiol 2007;213:374–83.

4. Levayer R, Lecuit T. Breaking down EMT. Nat Cell Biol 2008;10:757–9.5. Sommers CL, Heckford SE, Skerker JM, Worland P, Torri JA, Thomp-

son EW, et al. Loss of epithelial markers and acquisition of vimentinexpression in adriamycin- and vinblastine-resistant human breastcancer cell lines. Cancer Res 1992;52:5190–7.

6. Hay ED, Zuk A. Transformations between epithelium and mesen-chyme: normal, pathological, and experimentally induced. Am J Kid-ney Dis 1995;26:678–90.

7. Radisky DC. Epithelial–mesenchymal transition. J Cell Sci 2005;118:4325–6.

8. Kalluri R, Weinberg RA. The basics of epithelial–mesenchymal tran-sition. J Clin Invest 2009;119:1420–8.

9. Bates RC, Mercurio AM. The epithelial–mesenchymal transition(EMT) and colorectal cancer progression. Cancer Biol Ther 2005;4:365–70.

10. Iwatsuki M, Mimori K, Yokobori T, Ishi H, Beppu T, Nakamori S, et al.Epithelial–mesenchymal transition in cancer development and itsclinical significance. Cancer Sci 2010;101:293–9.

11. Joyce JA, Pollard JW. Microenvironmental regulation of metastasis.Nat Rev Cancer 2009;9:239–52.

12. Pollard JW. Tumour-educated macrophages promote tumour pro-gression and metastasis. Nat Rev Cancer 2004;4:71–8.

13. Condeelis J, Pollard JW.Macrophages: obligate partners for tumor cellmigration, invasion, and metastasis. Cell 2006;124:263–6.

14. Kaplan RN, Riba RD, Zacharoulis S, Bramley AH, Vincent L, Costa C,et al. VEGFR1-positive haematopoietic bone marrow progenitorsinitiate the pre-metastatic niche. Nature 2005;438:820–7.

15. Gao D, Nolan DJ, Mellick AS, Bambino K, McDonnell K, Mittal V.Endothelial progenitor cells control the angiogenic switch in mouselung metastasis. Science 2008;319:195–8.

16. ImmunoMembrane. Available from: http://imtmicroscope.uta.fi/immunomembrane/�.

17. Albini A, Sporn MB. The tumour microenvironment as a target forchemoprevention. Nat Rev Cancer 2007;7:139–47.

18. Arias AM. Epithelial–mesenchymal interactions in cancer and devel-opment. Cell 2001;105:425–31.

19. Hirohashi S. Inactivation of the E-cadherin-mediated cell adhesionsystem in human cancers. Am J Pathol 1998;153:333–9.

20. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57–70.

21. Gao D, Mittal V. The role of bone-marrow-derived cells in tumorgrowth, metastasis initiation and progression. Trends Mol Med2009;15:333–43.

22. Thiery JP, Acloque H, Huang RYJ, Nieto MA. Epithelial–mesen-chymal transitions in development and disease. Cell 2009;139:871–90.

23. Kang M, Jiang B, Xu B, Lu W, Guo Q, Xie Q, et al. Delta likeligand 4 induces impaired chemo-drug delivery and enhancedchemoresistance in pancreatic cancer. Cancer Lett 2013;330:11–21.

24. Sureban SM, May R, Mondalek FG, Qu D, Ponnurangam S,Pantazis P, et al. Nanoparticle-based delivery of siDCAMKL-1increases microRNA-144 and inhibits colorectal cancer tumorgrowth via a Notch-1 dependent mechanism. J Nanobiotechnol2011;9:40.

25. NamDH, JeonHM, KimS, KimMH, Lee YJ, LeeMS, et al. Activation ofNotch signaling in a xenograft model of brain metastasis. Clin CancerRes 2008;14:4059–66.

26. Reedijk M, Odorcic S, Chang L, Zhang H,Miller N, McCready DR, et al.High-level coexpression of JAG1 and NOTCH1 is observed in humanbreast cancer and is associated with poor overall survival. Cancer Res2005;65:8530–7.






27. Zhang Y, Li B, Ji ZZ, Zheng PS. Notch 1 regulates the growth of humancolon cancers. Cancer 2010;116:5207–18.

28. LeongKG,NiessenK, Kulic I, Raouf A, EavesC, Pollet I, et al. Jagged1-mediated Notch activation induces epithelial-to-mesenchymal transi-tion through Slug-induced repression of E-cadherin. J Exp Med 2007;204:2935–48.

29. Wang Z, Li Y, Kong D, Sarkar FH. The role of Notch signalingpathway in epithelial–mesenchymal transition (EMT) during devel-opment and tumor aggressiveness. Curr Drug Targets 2010;11:745–51.

30. Yang Y, Ahn YH, Gibbons DL, Zang Y, Lin W, Thilaganathan N, et al.TheNotch ligand Jagged2 promotes lung adenocarcinomametastasisthrough a miR-200-dependent pathway in mice. J Clin Invest 2011;121:1373–85.

31. PeinadoH, AleckovicM, Lavotshkin S,Matei I, Costa-Silva B,Moreno-BuenoG, et al. Melanoma exosomes educate bonemarrow progenitorcells toward a pro-metastatic phenotype through MET. Nat Med2012;18:883–91.

32. Xing F, Okuda H, Watabe M, Kobayashi A, Pai SK, Liu W, et al.Hypoxia-induced Jagged2 promotes breast cancer metastasis

and self-renewal of cancer stem-like cells. Oncogene 2011;30:4075–86.

33. IshiharaM, Nishida C, Tashiro Y, Gritli I, Rosenkvist J, Koizumi M, et al.Plasmin inhibitor reduces T-cell lymphoid tumor growth by suppres-sing matrix metalloproteinase-9-dependent CD11b(þ)/F4/80(þ) mye-loid cell recruitment. Leukemia 2012;26:332–9.

34. Ahn GO, Tseng D, Liao CH, Dorie MJ, Czechowicz A, Brown JM.Inhibition of Mac-1 (CD11b/CD18) enhances tumor response to radi-ation by reducing myeloid cell recruitment. Proc Natl Acad Sci U S A2010;107:8363–8.

35. Toh B, Wang X, Keeble J, Sim WJ, Khoo K, Wong WC, et al. Mesen-chymal transition and dissemination of cancer cells is driven bymyeloid-derived suppressor cells infiltrating the primary tumor. PLoSBiol 2011;9:e1001162.

36. Thurston G, Noguera-Troise I, Yancopoulos GD. The Delta paradox:DLL4 blockade leads to more tumour vessels but less tumour growth.Nat Rev Cancer 2007;7:327–31.

37. WangZ, Li Y, Banerjee S, Sarkar FH. Exploitation of the notch signalingpathway as a novel target for cancer therapy. Anticancer Res 2008;28:3621–30.

Caiado et al.





2013;73:4233-4246. Published OnlineFirst May 30, 2013.Cancer Res Francisco Caiado, Tânia Carvalho, Isadora Rosa, et al. Colorectal CancerEpithelial-to-Mesenchymal Transition and Metastasization in

Cells Promote+Jagged2+Derived CD11b−Bone Marrow

Updated version

10.1158/0008-5472.CAN-13-0085doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2013/05/30/0008-5472.CAN-13-0085.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/73/14/4233.full#ref-list-1

This article cites 36 articles, 8 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/73/14/4233.full#related-urls

This article has been cited by 1 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/73/14/4233To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-13-0085

http://cancerres.aacrjournals.org/content/suppl/2013/05/30/0008-5472.CAN-13-0085.DC1

http://cancerres.aacrjournals.org/content/73/14/4233.full#ref-list-1

http://cancerres.aacrjournals.org/content/73/14/4233.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/73/14/4233


bone marrow derived cd11bþjagged2þ cells promote...

Documents