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or and Stem Cell Biology

iprocal Activation of Prostate Cancer Cells and Cancer-ociated Fibroblasts Stimulates Epithelial-Mesenchymal

R

nsition and Cancer Stemness

Giannoni1, Francesca Bianchini2, Lorenzo Masieri3, Sergio Serni3, Eugenio Torre2,

alorini2, and Paola Chiarugi1

ractAlth

their fcarcintion. Inmesenmetas

driving indate, diffe

s' Affiliatioence, Tuser and Hiimental Pae, Florence

Supplemench Online (h

ponding Agy and On134 Firenz0; E-mail: lmical Scien, Italy. Phohiarugi@uni

.1158/0008-

American A

acrjourna

ough cancer-associated fibroblasts (CAF) are key determinants in the malignant progression of cancer,unctional contribution to this process is still unclear. Analysis of the mutual interplay between prostateoma cells and CAFs revealed a mandatory role of carcinoma-derived interleukin-6 in fibroblast activa-turn, activated fibroblasts through secretion of metalloproteinases elicit in cancer cells a clear epithelial-chymal transition (EMT), as well as enhancement of tumor growth and development of spontaneoustases. CAF-induced EMT leads prostate carcinoma cells to enhance expression of stem cell markers, ass the ability to form prostaspheres and to self-renew. Hence, the paracrine interplay between CAFs and

well a

cancer cells leads to an EMT-driven gain of cancer stem cell properties associated with aggressiveness andmetastatic spread. Cancer Res; 70(17); 6945–56. ©2010 AACR.

mainlotherfibrobchymafibrobThe

sary rtumor(10). Eior bynutrielate acontrobeen rtumorIn p

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duction

cinomas are the most frequent human malignant tu-and several lines of evidence support the notion thatowth and the invasive potential of carcinoma cells areced by host stromal cells, collectively called “reactivea” (1–3). Among stromal host cells, fibroblasts have beened to play a key role, mainly through secretion of solubles, as growth factors or inflammatory cytokines, as wellduction of extracellular matrix proteins and their pro-(2, 4). These activated fibroblasts are involved in creat-iche for cancer cells, promoting their motility. Indeed,-associated fibroblasts (CAF) show some degree of plas-controlled by tumor cells themselves, undergoing antiation process called mesenchymal-mesenchymalion (5, 6). Although the notion that CAFs acquire a phe-similar to myofibroblasts (MF) is widely accepted, the

vivo this transition are not yet fully elucidat-rentiation towardMFs can be induced in vitro

cells tbiologdeed,main uexpresCAFslial ceHow

whethto invas welular inachievmainldeed,acquirmigra

ns: 1Department of Biochemical Sciences, Universitycany Tumor Institute and “Center for Research,gh Education DenoTHE” ; and Departments ofthology and Oncology and 3Urology, University of, Italy

tary data for this article are available at Cancerttp://cancerres.aacrjournals.org/).

uthors: Lido Calorini, Department of Experimentalcology, University of Florence, Italy, Viale Morgagnie, Italy. Phone: 39-055-4598207; Fax: 39-055-ido.calorini@unifi.it or Paola Chiarugi, Department ofces, University of Florence, Viale Morgagni 50, 50134ne: 39-055-4598343; Fax: 39-055-4598905; E-mail:fi.it.

5472.CAN-10-0785

ssociation for Cancer Research.

ls.org

y by transforming growth factor-β (TGF-β), althoughgrowth factors have been reported to partially activatelasts (2). Activated fibroblasts express several mesen-l markers such as α-smooth muscle actin (α-SMA),last-activating protein (FAP), and vimentin (7).presence of activated fibroblasts seems to be a neces-equirement for growth and dissemination of severalcells, including prostate (8, 9) and breast carcinomaxtracellular matrix may also regulate tumor cell behav-facilitating cell contacts, angiogenesis, or transport ofnts. Moreover, CAFs secrete proteins that may stimu-dhesion, motility, and escaping from the local growthl, as well as de novo angiogenesis (2, 11). CAFs have alsoeported to exert a prometastatic effect for pancreaticcells (12).arallel to the ability of activated fibroblasts to stimulater progression toward an aggressive phenotype, tumorhemselves stimulate activated stromal cells to releaseical agents for their growth and dissemination (11). In-although the molecular players of such interactions re-nknown, prostate cancer epithelium stimulate CAFs tos markers characteristic of the MF phenotype (13), andcoinoculated with initiated but nontumorigenic epithe-lls promoted their in vivo tumor growth (14).ever, additional studies are needed to determineer CAFs represent a unique fibroblast phenotype andestigate the mechanism leading to fibroblast activationl as their contribution to tumor progression. A partic-terest would be devoted to the role of CAFs in theement of a motile/invasive phenotype of tumor cells,y through epithelial-mesenchymal transition (EMT). In-cancer cells undergoing EMT lose cell-cell contacts and

e a mesenchymal phenotype developing invasive andtory abilities, as well as developing stem-like properties

6945

(15–17tweenblast cbenigntify a(IL-6)metallcer stemation

Mate

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Giannoni et al.

Cance6946

). In this context, we studied the reciprocal interplay be-prostate carcinoma (PCa) cells and their stromal fibro-ounterpart. Using fibroblasts from human patients withprostatic hyperplasia or aggressive carcinoma, we iden-circuitry in which cancer cell–produced interleukin-6affects fibroblast activation, which in turn secretematrixoproteinases (MMP) eliciting EMT and enhancing can-

mness in PCa cells, thereby culminating in tumor for- or wit

Y2756or uPAtomiginvadiand th

MMPMed

ized w50 mmCaCl2in acegels wware (

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usingcells wplatewith Bfibrobgrowt15 toprostatasphedilutiowas pcell clocloneswere pCells wadhere

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nationof Aniform tand mmale(Charl

and spontaneous lung metastatic growth.

rials and Methods

ialsess specified, all reagents were obtained from Sigma.Cruz Biotechnology antibodies were as follows: FAP,, Vimentin, E-Cadherin, Snail, and Twist. Abcam anti-s were as follows: IL-6, cytokeratin, collagen I, and. BD Bioscience antibodies were as follows: CD44G44-26) and CD24 (clone ML5). Ilomastat, Y27632,

ho Kinase Inhibitor (n° 555551) were from Calbiochem.1 and IL-6 were from PeproTech. Urokinase-type plas-en activator receptor (uPAR)-blocking antibodies, n. 3936) were from American Diagnostica, Inc.

ulturean PCa cells (PC3, DU145, and LNCaP) and humante epithelial PNT-1A cells were from the European Col-of Cell Cultures. Human prostate fibroblasts [HPF,

ncer associated fibroblast (NAF), and CAF] were isolat-surgical explantation. Briefly, tissue samples from pa-

affected by benign prostatic hyperplasia or from healthyncerous regions of PCa-bearing patients (Gleason 4+5;AF, and CAF, respectively) were obtained asepticallyatients undergoing radical prostatectomy. Tissues wereed overnight in 1 mg/mL collagenase I, and cells werein DMEM containing 10% fetal bovine serum (FBS; alsoU145 cells, and prostate fibroblasts). LNCaP and PNT-1ere cultured in RPMI 1640 added with 10% FBS. Condi-media (CM) were obtained by 48-hour serum-starvedlarified by centrifugation, and used freshly.

blast activations were grown to subconfluence and treated for 24 hour0 ng/mL rTGF-β1, 50 ng/mL IL-6, or CMPC3. Fresh-free medium was added for an additional 24 hours be-llection of CM.

noprecipitation and Western blot analysisoblasts or PC3 cells (1 × 106) derived fromour experimen-ditions were lysed on ice in radioimmunoprecipitationbuffer [50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl,ton X-100, 2 mmol/L EGTA, 1 mmol/L sodium orthova-, 1 mmol/L phenylmethane sulfonyl-fluoride, 10 μg/mLnin, and 10μg/mL leupeptin]. Twenty μg of total proteinsoaded on SDS-PAGE, separated, and transferred onto

ellulose. The immunoblots were incubated in 3% bovinealbumin, 10 mmol/L Tris-HCl (pH 7.5), 1 mmol/L EDTA,

with 1For co

r Res; 70(17) September 1, 2010

1% Tween 20 at room temperature and probed with pri-nd appropriate secondary antibodies.

ro Boyden invasion assayt-micrometer Transwells were coated with 50 μg/cm2

nstituted Matrigel. PC3 cells (5 × 104) were loaded intoper compartment in serum-free growth medium, withhout Ilomastat (50 μmol/L), Aprotinin (100 μg/mL),3 (10 μmol/L), the Rho Kinase inhibitor (10 μmol/L),R-blocking antibodies (3 μg/mL). Cells were allowedrate toward complete growthmedium for 24 hours; non-ng cells were removed mechanically using cotton swabs;e membrane was stained with DiffQuick solution.

zymographyia were electrophoresed on 8% SDS-PAGE copolymer-ith 0.1% (w/v) type A gelatin. Gels were incubated inol/L Tris-HCl (pH 7.4), 200 mmol/L NaCl, and 5 mmol/Lat 37°C for 24 hours, stained with 0.1% Coomassie bluetic acid, methanol, and distilled water (1:2:3). Destainedere scanned with Quantity-One Image Analysis soft-Bio-Rad).

asphere formation and clonogenicity assaycells incubated for 72 hours with CM were detachedAccutase (Sigma). For prostasphere formation, singleere plated at 150 cells/cm2 on low-attachment 100-mm(Corning) in DMEM/F12 (Invitrogen) supplemented27 and N2 (Invitrogen), 5 μg/mL insulin, 20 ng/mL basiclast growth factor (FGF) and 20 ng/mL epidermalh factor. Cells were grown under these conditions for20 days and formed nonadherent P0 spheres termedspheres. For the evaluation of self-renewal, a single pros-re was dissociated in single cells with Accutase and an of one cell per well into 96-well low-attachment plateserformed to isolate individual P1 prostaspheres. Single-ning was confirmed by microscopic analysis, and singlewere counted. For clonogenicity assay, 8 × 102 PC3 cellslated in 35-mm culture dishes in complete DMEM/F12.ere grown under these conditions for 15 to 20 days, andnt separated clones were counted.

cytometrycells (1 × 106) were labeled with FITC-anti-CD44G44-26) and PE-anti-CD24 (clone ML5) antibodiesour at 4°C in the dark. Cells were washed and flow cy-ry was performed using a FACSscan (BD Biosciences).

raft experimentsivo experiments were conducted in accordance withal guidelines and approved by the ethical committeemal Welfare Office of Italian Work Ministry and con-o the legal mandates and Italian guidelines for the careaintenance of laboratory animals. Six- to 8-week-oldsevere combined immunodeficient (SCID)-bg/bg micees River Laboratories International) were injected s.c.

× 106 PC3 cells, both in the right and left lateral flanks.injection with fibroblasts, 1 × 106 PC3 cells and 0.5 × 106

Cancer Research

of fibrand inmonitby a c(L) anorganfor his

ImmuFor

into 5chargedratedstain.formecubatTritonat 4°Cnostai(PicTuwith hthroug

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oblasts were combined, resuspended in 100 μL of PBS,jected s.c. in mice. Animals (four to six per group) wereored daily; tumor size was measured every 2 to 3 daysaliper; tumor volumes were determined by the lengthd the width (W): V = (LW2)/2 (18). Tumors and others were fixed overnight at 4°C in formalin (5% in PBS)tologic analyses.

nohistochemical analysismalin-fixed, paraffin-embedded tissue blocks were cutμm consecutive sections and mounted on positivelyd slides. Tissue sections were deparaffinazed and rehy-before staining with H&E or Gomori silver trichromicSome sections were used for antigen retrieval, per-d for 20 minutes with citrate buffer (pH 6.0), before in-ion in a solution (LaionbVisreion) containing 0.1%X-100. These sections were then incubated overnightwith the primary antibody of interest and then immu-ning was carried out using a commercially available kitre Plus kit, Zymed). Slides were then counterstainedematoxylin. Positive and negative controls were usedhout all immunostaining protocols.

tical analysisa are presented as means ± SD from at least three in-dent experiments. Statistical analysis of the data wasmed by Student's t test. P values of ≤0.05 were consid-tatistically significant.

lts

ted fibroblasts induce invasiveness in PCa cellsanalyze the effect of fibroblasts of tumor stroma onrogression, we selected as a model PC3 cells, a lined from a bone metastasis of human PCa cells. Organ-ic fibroblasts were isolated from prostate removedpatients affected by benign prostatic hyperplasianalyzed for their in vivo and in vitro activation. Ex-on of E-cadherin and cytokeratin was performedlude epithelial contamination (Supplementary Fig.We compared fibroblast activation achieved throughnventional TGF-β treatment, named MFs (2), andposure to PC3-CM, named PCa-activated fibroblastsF; Fig. 1A). We observed a similar behavior of bothents in enhancing fibroblast motility and collagen Iction (Supplementary Fig. S1B and C). However,wo treatments revealed differential expressionknowledged markers of fibroblast activation asor FAP (Fig. 1B; refs. 2, 6), suggesting that PC3 cellsfibroblasts differently from the simple stimulationGF-β.evaluate the effect of MFs and PCa-AFs on PCa aggres-ss, we analyzed both activation of the EMT processree-dimensional matrix invasiveness in PC3 cells incu-with CM by MFs and PCa-AFs. EMT has already beenated with PCa progression and the achievement of a

asive behavior, and TGF-β has been specifically in-(8). The analysis of cell morphology, as well as the

liferatthe na

acrjournals.org

sion of known EMT markers (19), as upregulation ofTwist, vimentin, and the Met and downregulation ofherin, revealed that both MFs and PCa-AFs are ableit differentiation toward a mesenchymal phenotypeC) as well as a proinvasive effect on PC3 cells (Fig. 1D),ting that both MFs and PCa-AFs may be active stromalnents of PCa progression.correlate PCa aggressiveness and fibroblast activation,mpared PC3, DU145, and LNCaP cells reported to pos-ecreasing aggressiveness both in vivo and in vitro (20)ir ability to stimulate fibroblast activation and therebyse PC3 invasiveness (Supplementary Fig. S2). The re-eveal that the ability to activate fibroblasts is sharedth PC3 and DU145, whereas LNCaP cells seem unableit this effect, suggesting a positive correlation betweenlast activation and PCa aggressiveness.reover, the ability of HPFs to increase three-dimensionalty and activate EMT is strictly limited to tumor cells., we observed that nontransformed epithelial PNT-1re not able to activate HPFs (Supplementary Fig. S3A).ping with our findings, HPFs treated with CM fromare not able to increase invasiveness of PC3 cells

lementary Fig. S3B). Moreover, PNT-1 cells do not re-to factors secreted by activated fibroblasts as well asct their EMT markers (Supplementary Fig. S3C and D).

ral IL-6–dependent generation of PCa-AFsanalyze the nature of signals originating by PC3 cells tofibroblast differentiation, we focused our attention onndeed, several reports indicate that this proinflamma-ytokine is highly produced by aggressive PCa, as well as3 and DU145 cell lines, whereas its production is low ort in nonaggressive PCa and in LNCaP cells (21). We con-that IL-6 is highly produced by both PC3 and DU145hereas it is absent in nontransformed PNT-1 cells and

g HPFs and slightly secreted by LNCaP, as well as bynd PCa-AFs (Supplementary Fig. S4; Fig. 2A). We there-sayed the sensitivity of HPFs to IL-6 and observed thateatment produces an activation of these fibroblasts inof their ability to increase PC3 invasiveness (Fig. 2B).theless, and in keeping with our previous observations,eatment of HPFs induces a strong increase in FAP butα-SMA expression (Fig. 2C), as well as an increase inine production of IL-6 (Supplementary Fig. S4). The keyIL-6–mediated signaling in eliciting HPF activation to-differentiated state affecting tumor cell invasiveness is

ore confirmed by treatment of HPFs with IL-6–blockingdies during their exposure to PC3 CM (Fig. 2D). In con-IL-6–blocking antibodies are unable to affect thecement in invasiveness induced by MFs.

Fs promote EMT and invasiveness in PC3 cellsgh MMPsivated fibroblasts have been reported to affect tumorancy mainly through secretion of cytokines, chemo-and growth factors, thereby enhancing cancer cell pro-

ion, survival, and invasive properties (22). To analyzeture of factors secreted by both MFs and PCa-AFs,

Cancer Res; 70(17) September 1, 2010 6947

and acorresfore reCM inthat, amote Pmoatt

focusebeen rcancezymogcells o

Figureor CMP

CM froD, PC3*, P < 0

Giannoni et al.

Cance6948

ffecting the phenotype of PC3 cells, we analyzed theponding CM for their chemoattracting effect. We there-peated the three-dimensional invasiveness assay usingthe lower chamber of a Boyden assay. The results showlthough both CM from MFs and PCa-AFs similarly pro-

invasion for 24 h toward complete growth medium. Photographs are repr.001 versus St Med.

C3 cell invasiveness, only CM from MFs displays a che-ractive effect (Fig. 3A). Our attention was therefore

a stroappea

r Res; 70(17) September 1, 2010

d on MMPs, nonchemoattracting agents, which haveeported to affect cell invasiveness and EMT of severalr models (2, 23, 24). The analysis of MMPs by gelatinraphy reveals that exposure of HPFs to CM from PC3r to IL-6 treatment, but not to TGF-β treatment, elicits

tive of six randomly chosen fields; columns, mean values.

1. Activated HPFs promote EMT and enhance PC3 invasiveness. A, experimental design. B, HPFs were activated for 24 h with 10 ng/mL TGF-β1C3, and serum-starved for 24 h. α-SMA and FAP expression was assessed by immunoblot analysis. C, PC3 cells were incubated for 48 h withm HPFs, MFs, and PCa-AFs. Top, photographs. Expression of vimentin, Met, E-Cadherin, Snail, and Twist was analyzed by immunoblotting.

esenta

ng increase in MMP-2 expression and the de novorance of MMP-9 (Fig. 3B). In addition, treatment of HPFs

Cancer Research

with Ifrom(Fig. 3MMP

EMT pPCa-A

FigurePC3 ceevaluat24 h wactivatethe corversus

CAF-Mediated EMT and Stemness

www.a

L-6–blocking antibodies during their exposure to CMPC3 abrogates the upregulation of MMP expression

responding CM, was analyzed. Photographs and bar graph, representative of sixcontrol CM PCa-AFs.

acrjournals.org

rocess (Fig. 3C) and the proinvasive effect elicited byFs on PC3 cells (Fig. 3D). In contrast, the EMT induc-

B). In keeping with our findings, we observed that theinhibitor ilomastat reverts both the activation of the

tion and the increase in PC3 invasiveness induced by MFs areinsensitive to MMP inhibition (Fig. 3C and D).

2. IL-6 is responsible for HPF activation induced by CMPC3. A, IL-6 was evaluated by immunoblotting on CM of PNT-1, LNCaP, DU145, orlls. B, PC3 cells were incubated with CM from HPFs; HPFs were stimulated with 50 ng/mL IL-6, MFs, and PCa-AFs; and their invasion wased. Photographs and bar graph, representative of six randomly chosen fields. *, P < 0.001 versus St Med. C, subconfluent HPFs were activated forith 10 ng/mL TGF-β1 (MFs), CMPC3 (PCa-AFs), or 50 ng/mL IL-6. α-SMA and FAP expression was assessed by immunoblotting. D, HPFs wered for 24 h with 10 ng/mL TGF-β1 (MFs) and CMPC3 (PCa-AFs) in the presence of IL-6–blocking antibodies. Invasion of PC3 cells, treated with

randomly chosen fields. *, P < 0.001 versus St Med. #, P < 0.001

Cancer Res; 70(17) September 1, 2010 6949

MFs amigraTo d

style s

surrouof PC

Figureanalyzeantibodwith CManalyzerandom CM PC

Giannoni et al.

Cance6950

nd PCa-AFs stimulate a proteolysis-basedtion in PC3 cells

from HPFs, MFs, and PCa-AFs in the presence of 50 μmol/L ilomastat,d by immunoblotting. D, PC3 cells were treated as in C, and their invasioly chosen fields. *, P < 0.001 versus St Med. #, P < 0.001 versus control

ate, EMT has been correlated with a polarized motilitytrongly involving proteolytic degradation of the matrix

themThe a

r Res; 70(17) September 1, 2010

nding the tumor (15, 19). To analyze the motility style3 upon exposure to activated fibroblasts, we treated

e expression of E-Cadherin, Met, vimentin, Snail and Twist wasevaluated. Photographs and bar graph, representative of sixa-AFs.

3. MP production by PCa-AFs leads to PC3 increased invasiveness. A, invasion of PC3 cells, toward CM from HPFs, MFs, or PCa-AFs, wasd. B, HPFs were activated for 24 h with 10 ng/mL TGF-β1 (MFs), 50 ng/mL IL-6 (HPFs+IL-6), or CMPC3 (PCa-AFs) with or without IL-6–blockingies, and then starved for 24 h. Gelatin zymography analysis was performed on the corresponding CM. C, PC3 cells were incubated for 24 h

and thn was

with two selective inhibitors of the small GTPase RhoA.im was to exclude a Rho-mediated motility style,

Cancer Research

commEMT,contraof ma

PCa-ANext,

Figurestrategfrom HPROCKB, PC3during iaprotinrepreseSt MedpreconHPFs +without

CAF-Mediated EMT and Stemness

www.a

only referred to as ameboid style, incompatible withreportedly based on Rho-dependent cytoplasmic

MMPsuPARspursystemmainlSelectsystemconfirexpres

AnalyFibr

prostaand hEpithecludedtary Fcarcin(Fig.to botactivasuggestimuvation(Fig.in vivotheir

CAF-mPC3 cTo

mor gof disthat ltifiedcondiweentumortion oresultFig. 5a simPC3 cicantlyhand,that tTumoreticu(Fig. 5cinomnote,animatastasthat tductiotype i

50 ng/mL IL-6 (HPFs+IL-6), or HPFs + CM (PCa-AFs) with orIL-6–blocking antibodies.

acrjournals.org

Fs stimulate a Rho-independent motility style (Fig. 4A).we treated PC3 cells with ilomastat, to selectively block, or with uPAR-blocking antibodies, to block uPA/proteases. The results reveal that MF-induced motilityis mainly mediated by the uPA/uPAR proteolytic, whereas PCa-AFs induce in PC3 cells a motility

y based on MMP degradation of barriers (Fig. 4B).ive involvement of uPA/uPAR or MMP proteolytics in MF or PCa-AF proinvasive effects has been

med with aprotinin treatment and analysis of MMPsion (Fig. 4C).

sis of CAFs from PCa specimensoblasts were obtained from the surgically explantedte of PCa-bearing patients (Gleason 4+5) of cancerealthy regions, called CAFs and NAFs, respectively.lial cell contamination of CAFs and NAFs was ex-by E-cadherin and cytokeratin analysis (Supplemen-

ig. S1A). We first observed that prostate CAFs fromoma are strongly positive to both α-SMA and FAP5A). Conversely, both HPFs and NAFs are negativeh markers. In addition, CAFs do not need an in vitrotion step to elicit a proinvasive effect on PC3 cells,sting that they have already received the activationlus in vivo (Fig. 5B). HPFs and NAFs need a preacti-step with IL-6 or TGF-β to elicit a similar effect

5B). In addition CAFs, although already activated, are still sensitive to IL-6, thereby further increasingproinvasive skill on PC3 cells (Fig. 5B).

ediated tumor formation and lung metastases ofellsshow that CAFs are a necessary requirement for tu-rowth and, eventually, for stimulating developmenttant metastases, we used PC3 cells in conditionsimited their in vivo growth. Pilot experiments iden-1 × 106 cells and absence of Matrigel as limitingtions. Subcutaneous tumors from the mixture bet-PC3 cells and prostate CAFs show a high rate ofgrowth, as well as tumor incidence, whereas injec-f PC3 cells with HPFs nonactivated in vitro do notin subcutaneous tumors at the end point (92 d;

C). CAFs, PCa-AFs, and IL-6–stimulated HPFs causeilar behavior. In contrast, tumor-forming ability ofells injected with NAFs is characterized by a signif-longer latency and reduced incidence. On the otherHPFs did not stimulate tumor growth, suggestinghese types of fibroblasts express a resting phenotype.rs, independently from their origin, express finelar fibers in tight contact with carcinoma cellsD). In addition, we detected several emboli of car-a cells inside peripheral venules (Fig. 5D, arrow). Ofhistologic examination of lungs of all tumor-bearingls revealed the presence of spontaneous microme-es of carcinoma cells (Fig. 5D), thereby confirminghe spur elicited by activated fibroblasts through in-

n of EMT gives rise to a highly aggressive pheno-

ction and independent from protelytic degradationtrix (25). The results indicate that both MFs and

4. MFs and PCa-AFs induce different proteolytic motilityies in PC3 cells. A, PC3 cells were incubated for 24 h with CMFs, MFs, and PCa-AFs in the presence of Y27632 (10 μmol/L) or

inhibitor (10 μmol/L), and PC3 cell invasion was evaluated.cells were treated as in A, except that invasion was evaluatednhibition of MMPs (ilomastat, 50 μmol/L) or uPA/uPAR (100 μg/mLin or 3 μg/mL uPAR-blocking antibodies). Bar graphs,ntative of six randomly chosen fields (B and C). *, P < 0.001 versus. C, gelatin zymography analysis was performed on PC3 cellsditioned with CM from HPFs, HPFs + 10 ng/mL TGF-β1 (MFs),

PC3

n PC3 cells.

Cancer Res; 70(17) September 1, 2010 6951

CAF-mstemRec

tion o

by extheref

Figurefor 24 hactivateof PC3primary ; originbg/bg-m

Giannoni et al.

Cance6952

ediated EMT promotes the generation of cancercells

tumor (top; original magnification, ×20) and lung micrometastases (bottomice were stained with H&E or with Gomori.

ently, Mani and colleagues (16) described the genera-f breast cancer stem cells driven by EMT induction

motepossib

r Res; 70(17) September 1, 2010

pression of Snail or Twist transcription factors. Weore speculated that CAF-stimulated EMT could pro-

al magnification, ×40) obtained by PC3/CAFs coinjection in SCID

5. CAFs from PCa strongly enhance the aggressiveness of PC3 cells. A, α-SMA and FAP expression analysis of HPFs, NAFs, or CAFs treatedwith 10 ng/mL TGF-β1 or 50 ng/mL IL-6 was performed by immunoblotting. B, PC3 cells were incubated with CM from HPFs, NAFs, or CAFsd as described in A, and their invasion was evaluated. Bar graph is shown. *, P < 0.001 versus St Med. C, xenograft growth in SCID bg/bg micecells s.c. injected with activated fibroblasts. Top, primary tumor growth; bottom, tumor incidence. D, paraffin-embedded tissue sections from

the generation of cancer stem cells. To address thisility, we induced an EMT in PC3 cells using CAFs or

Cancer Research

HPFscity anpressi27). In

gone aulated

FigureTGF-β1Left, PCSt. Medby meaMed. Cimmunofibrobla

CAF-Mediated EMT and Stemness

www.a

stimulated by IL-6 or TGF-β and analyzed clonogeni-d self-renewal ability of PC3 cells, as well as the ex-

on of acknowledged cancer stem cell markers (16, 26, forma

sts and PCa cells through secretion of IL-6 (efferent pathway) and MMP-depend

acrjournals.org

n EMT following treatment with CAFs or HPFs stim-by IL-6 or TGF-β show a 4- to 5-fold increase in the

tion of nonadherent prostaspheres, a property associ-

terestingly, we observed that the cells that had under- ated with prostate stem cells (Fig. 6A, left). EMT driven by

6. CAFs, PCa-AFs, and MFs induce cancer cell stemness. A, right, PC3 cells were incubated with CM from HPFs, stimulated with 10 ng/mLor 50 ng/mL IL-6, and CAFs. P1 individual spheres derived from PC3 cells were counted, photographed, and plotted. *, P < 0.001 versus St. Med.3 cells treated as above were assayed for clonogenicity in adherent cultures. Separated clones were counted and plotted. *, P < 0.001 versus. B, PC3 cells treated as in A were analyzed for expression of the cell surface marker FITC-CD133 (left) or FITC-CD44 and PE-CD24 (right)ns of fluorescence-activated cell sorting analysis. The CD133-positive or CD44high/CD24low populations were plotted. *, P < 0.001 versus St., paraffin-embedded tissue sections from xenografts and lung micrometastases obtained by PC3/CAFs coinjection in SCID bg/bg-mice werestained with CD44 and CD24 antibodies. Representative images are shown (original magnification, ×40). D, reciprocal interplay between stromal

ent EMT in PCa cells (afferent pathway).

Cancer Res; 70(17) September 1, 2010 6953

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or activated HPFs leads to increase in PC3 clonogeni-s assessed by their ability to form colonies followingg dilution (Fig. 6A, right). Ilomastat administrationts P1 prostasphere formation by HPF + IL-6 or CAF-d PC3, whereas it is almost ineffective on MF-elicitedspheres (Supplementary Fig. S5). In keeping with ourgs with the behavior observed in breast and prostater stem cells (16, 26), PC3 cells that underwent anfollowing exposure to CAFs or activated HPFs showrease in the number of CD133-positive cells andigh/CD24low cells (Fig. 6B). In particular, cytofluori-analysis revealed that 65% of nontreated PC3 cells44high and that EMT after CAF treatment leads torease in this population to 99%. Conversely, inductionT by CAFs is associated with progressive increase ofow cells, which account for 15% of wild-type PC3 cells0% of cells undergoing EMT. In addition, the analysisor-forming ability of PC3 cells, using a limiting cellntration, revealed an enhancement of tumor-formingof PC3 cells coinjected with CAFs or activated HPFsC). Interestingly, the analysis of both xenografts andneous lung metastases from CAF- or activated HPF-ated cells confirms that these tumor-repopulating cellstally CD24 negative and highly CD44 positive (Fig. 6C).en together, these observations suggest that the acti-of specific signal transduction pathways elicited byFs helps to generate a population of prostate cancerells with defined ability to form primary tumors andt metastases (Fig. 6D).

ssion

ing prostate cancer progression, reactive stroma col-tes to disease progression allowing or initiatingnisms promoting the escape of tumor cells from im-surveillance or hormonal control and to achieve a mo-enotype useful to colonize distant organs (2, 11, 28).losely interactive pathways are established in thetalk between cancer and stromal cells: (a) in the “effer-athway, cancer cells trigger a reactive response in thea, and (b) in the “afferent” pathway, the modified stro-lls in the surrounding microenvironment affect cancersponses (11). With respect to the efferent pathways,few cytokines have been reported to be released bycells and affect differentiation of fibroblasts. An ac-

edged role is played by TGF-β1 and TGF-β2, stronglyin eliciting the MF phenotype, whereas both platelet-d growth factor or FGF-2 are only active in promotinglast proliferation but not differentiation (2, 29). Wereport that IL-6, produced by PC3 cells, is able to eliciticular fibroblast activated phenotype (named PCa-AFs).Fs do not express α-SMA, typical of MFs, but their ac-state is confirmed by enhanced expression of FAP

ecretion of extracellular matrix. Of note, PCa-AFsly activate the EMT process and invasiveness in PC3Abrogation of IL-6–mediated cross-talk between PCa

nd fibroblasts leads to the elimination of inductionT and invasiveness. IL-6 production has already been

matricthe co

r Res; 70(17) September 1, 2010

ed in cancer cells, and its levels have been correlatedarcinoma aggressiveness (8, 30, 31). In keeping withdings, both highly aggressive PC3 and DU145 cells pro-high level of IL-6, whereas the less aggressive LNCaPo not. It is possible that the PCa-AF IL-6–dependenttype is either an intermediate state of activation duringfferentiation or an independent and terminal activation

afferent way is represented by the effect exerted byted fibroblasts on cancer cell evolution toward an ag-e phenotype. The number of known cytokines releaseds or CAFs is large: hepatocyte growth factor, vascularhelial growth factor, FGF-2, insulin-like growth factor-1,-6 (5, 7). We now report that PCa-AFs and MFs secretelevel of IL-6 and a large amount of MMPs, which aresible for inducing a clear EMT in PC3 cells. In keepingur findings, production of MMP-2 and MMP-9, as well6 levels, have been associated with fibroblast senes-, a phenotype of stromal fibroblasts involved in pros-arcinogenesis and tumor progression (32, 33).observed that CAFs, isolated from human aggressivere sensitive to in vitro IL-6 stimulation, and their re-e is very similar to PCa-AFs in terms of EMT and inva-ss elicited in PC3 cells. Therefore, it is conceivable thatthe population of CAFs, which escort the tumor mass,posed of multiple phenotypes, including MFs andensitive fibroblasts. Indeed, we found among CAFs apopulation that respond to both IL-6 and TGF-β, sug-g coexistence of phenotypes resembling MFs and PCa-e can speculate that different CAF populations candifferent steps of tumor development, as well as thatnd late phases of cancer progression may have differ-fects on the activation of intratumoral fibroblasts. Thisfurther confirmed by the absence of EMT in nontrans-d prostate epithelial cells or by the inability of LNCaPo activate fibroblasts. In keeping with our findings, pre-data indicate a differential role of activated stroma onl or initiated epithelial prostate cells in the stimulationivo tumorigenesis (14, 34, 35).report herein that CAF stimulation results in a clearf cancer cells, which acquire a proteolysis-based motil-well as short tumor latency, high rate of tumor growth,cidence. The spur given by CAF coinjection is extreme-erful as it elicits spontaneous lung metastases of tumoro our knowledge, this is the first observation of spon-s metastases of human PCa cells injected in a hetero-site in mouse models. In keeping with our findings,chymal stem cells, a pluripotent progenitor populationapacity to differentiate into fibroblasts (36), facilitatetatic spread of breast carcinoma cells (37).T, critical for appropriate embryonic development,healing, tissue regeneration, and organ fibrosis, has

een involved in cancer progression, enhancing mesen-l features, reducing cell-cell contact, and increasingty (15, 19, 25). EMT undergoing cells usually movegh proteolytic degradation of three-dimensional

es and creating a path to invade it. We observed thatnditioning of PC3 cells with MFs or PCa-AFs gives rise

Cancer Research

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asiveness spurs involving different protease systems,, uPA/uPAR for MFs, and MMPs for PCa-AFs. It is like-t this differential involvement of proteases should re-different state of activated fibroblast subpopulationsed by a multiplicity of signals originating from tu-ells.ently, EMT has been classified as type 1 EMT involvedryonic development, type 2 in tissue repair, and type 3tastatic spreading of cancer (15). Type 2 and 3 EMTtheir dependence from inflammation and are charac-from their endurance until the provoking spur is re-. Our findings reveal that CAF-mediated EMT in PC3hare with type 3 EMT the decrease of E-cadherin;se in Snail, Twist, vimentin, and Met; and the use oflysis-based invasiveness. Interestingly, CAF-mediatedeculiarity is to be engaged by MMPs secreted by acti-fibroblasts. To our knowledge, only two reports indi-forced expression or exogenous addition of MMP-3ilitate EMT (23, 38). In addition, MMP-2 and MMP-9een shown to cleave E-cadherin, which leads to its in-ization and to relocalization of transcriptionally activenin to the nucleus, inducing EMT (2, 24). Type 3 EMTlitated by genomic and epigenetic alterations acquiredcer cells, and some of these alterations have been re-also in the associated stroma. In keeping with our

gs, we found that NAFs are able to cause primary tu-although with a delay with respect to CAFs. Converse-Fs are completely unable to promote tumor growth.nding suggests that in vivo prostate fibroblasts shouldgo genetic or epigenetic alterations due to malignantmodeling of environment. Although in the literature,at present a controversial point, some reports haved that p53 mutations or loss of heterozigosity are com-vents in cancer stromal cells (39, 40).T has been recently reported to generate cells with can-em cells properties in breast and prostate cancers ass in nontransformed cells (41). CD44high/CD24low stemxpress typical EMT markers and, conversely, cells com-to EMT by forced expression of Snail or Twist un-

a dramatic enhancement of expression of stemnessrs (16, 26, 41). Furthermore, CD44high/CD24low cancer

ReceOnlineF

z B, Phan SH, Thannickal VJ, Galli A, Bochaton-Piallat ML,bbiani G. The myofibroblast: one function, multiple origins. Am Jthol 2007;170:1807–16.

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cells are more tumorigenic in vivo. Nevertheless, theseutstandingly introduce EMT to the most importante for a cancer cell, that is, the ability to self-renewegenerate the original cancer, though the nature ofnals that prompt EMT-driven cancer stemness in vivoned obscure. Herein, we show that the diabolic circuitryen stromal fibroblasts and PCa cells leads to an EMT-dent increase in CD44high/CD24low-ratio and CD133ssion, as well as to enhancement of clonogenicity,newal, and tumorigenic properties of PCa. IL-6 is aful activating signal for stromal fibroblasts, as indicat-its ability to mimic the complexity of stimuli actingon CAFs. In addition, prostate cancer cells undergoingediated EMT generate CD44high/CD24low xenograftsore important, spontaneous lung metastases. The lat-ult is extremely provocative as it supports the notionlthough EMT plays a role in the generation of high-invasive cells with cancer stem cell–like properties,me stem cell population is responsible for metastaticination. Therefore, we propose a correlation betweenndergoing CAF-mediated EMT and cancer stemness inof their ability to spread and reconstitute metastatics. In this context, cancer stemness, and by extensiontatic dissemination, is directly induced by fibroblaststumor microenvironment. Control or suppression ofediated EMT may serve as a basis for the developmentrapies that target tumor growth and dissemination indary organs.

osure of Potential Conflicts of Interest

otential conflicts of interest were disclosed.

Support

ciazione Italiana Ricerca sul Cancro, by Istituto Toscano Tumori, andione Cassa di Risparmio di Lucca.costs of publication of this article were defrayed in part by the paymentcharges. This article must therefore be hereby marked advertisement innce with 18 U.S.C. Section 1734 solely to indicate this fact.

ived 03/05/2010; revised 06/10/2010; accepted 06/27/2010; publishedirst 07/20/2010.

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