Stromal Fibroblasts Present in Invasive Human Breast Carcinomas

Cell,Vol.121,335–348,May6,2005,Copyright?2005by Elsevier Inc.DOI10.1016/j.cell.2005.02.034

Stromal Fibroblasts Present in Invasive Human Breast Carcinomas Promote Tumor Growth and Angiogenesis through Elevated SDF-1/CXCL12Secretion

Akira Orimo,1Piyush B.Gupta,1,2Dennis C.Sgroi,4Introduction

Fernando Arenzana-Seisdedos,7Thierry Delaunay,8

Rizwan Naeem,6Vincent J.Carey,5Neoplastic epithelial cells coexist in carcinomas with Andrea L.Richardson,3and Robert A.Weinberg1,2,*

several distinct stromal cell types that together create 1Whitehead Institute for Biomedical Research the microenvironment of the cancer cells.The contribu-

tion of the stromal microenvironment to the develop-Cambridge,Massachusetts02142

2Department of Biology ment of a wide variety of tumors has been supported by Massachusetts Institute of Technology

extensive clinical evidence(Coussens and Werb,2002; Cambridge,Massachusetts02139Jacobs et al.,1999)and by the use of experimental 3Department of Pathology

mouse models of cancer pathogenesis(Barcellos-Hoff The Brigham and Women’s Hospital and Ravani,2000;Bhowmick et al.,2004a;Elenbaas et Harvard Medical School

al.,2001;Sieweke et al.,1990).The accumulated evi-Boston,Massachusetts02115dence indicates that tumor cells actively recruit stromal 4Department of Pathology

cells,such as inflammatory cells,vascular cells,and Harvard Medical School fibroblasts(Bhowmick et al.,2004b;Cunha et al.,2003;

Olumi et al.,1999;Tlsty,2001),into the tumor,and that Molecular Pathology Research Unit

Massachusetts General Hospital this recruitment is essential for the generation of a mi-Boston,Massachusetts02129

croenvironment that actively fosters tumor growth.

5Channing Laboratory The presence of large numbers of myofibroblasts, Harvard Medical School

which expressα-smooth muscle actin,is apparent in Boston,Massachusetts02115the stromal compartment of most invasive human 6Texas Children’s Cancer Center

breast cancers(Sappino et al.,1988).Such myofi-Department of Pediatrics and Pathology broblasts,sometimes termed“activated fibroblasts,”Baylor College of Medicine

are also known to be present in areas of inflammation Houston,Texas77030and in tissues undergoing the remodeling seen during 7Unitéd’Immunologie Virale

wound healing(Mueller and Fusenig,2004;Serini and Institute Pasteur Gabbiani,1999).Various types of“reactive”or“des-Paris75724

moplastic”tumor stroma generate granulation tissue, France which is composed of myofibroblasts as well as large 8INRA

amounts of accumulated extracellular matrix(Bissell Villenave d’Ornon33883and Radisky,2001).Desmoplastic stroma is often found France

in commonly occurring epithelial malignancies,includ-

ing those of the breast,prostate,colon,lung,and

uterus(Orimo et al.,2001).

Summary Previous reports indicate poorer prognoses associ-

ated with carcinomas bearing desmoplastic stroma Fibroblasts often constitute the majority of the stro-(Cardone et al.,1997;Maeshima et al.,2002).Stromal mal cells within a breast carcinoma,yet the functional myofibroblasts extracted from human breast carcino-contributions of these cells to tumorigenesis are mas also exhibit distinctive gene-expression profiles poorly https://www.360docs.net/doc/066463194.html,ing a coimplantation tumor xe-(Allinen et al.,2004),and fibroblasts explanted from hu-nograft model,we demonstrate that carcinoma-asso-man prostate carcinomas can assist tumor formation ciated fibroblasts(CAFs)extracted from human breast(Olumi et al.,1999).These reports suggest that fibro-carcinomas promote the growth of admixed breast car-blasts in tumor masses possess biological characteris-cinoma cells significantly more than do normal mam-tics distinct from those of normal fibroblasts.However, mary fibroblasts derived from the same patients.The the precise functional contributions of the fibroblasts CAFs,which exhibit the traits of myofibroblasts,play within stroma to carcinoma growth and progression re-a central role in promoting the growth of tumor cells main poorly understood at the biochemical and cellular through their ability to secrete stromal cell-derived level.In the present study,we sought to elucidate the factor1(SDF-1);CAFs promote angiogenesis by re-properties of fibroblasts isolated from invasive human cruiting endothelial progenitor cells(EPCs)into carci-mammary carcinomas that facilitate tumorigenesis. nomas,an effect mediated in part by SDF-1.CAF-

secreted SDF-1also stimulates tumor growth directly,Results

acting through the cognate receptor,CXCR4,which is

expressed by carcinoma cells.Our findings indicate Isolation of Primary Fibroblastic Population

that fibroblasts within invasive breast carcinomas from Invasive Human Breast Cancers

contribute to tumor promotion in large part through We extracted fibroblasts from six human invasive mam-the secretion of SDF-1.mary ductal carcinomas obtained from mastectomies.

The tumor masses were dissociated,and various cell

types were separated to obtain populations of carci-*Correspondence:weinberg@https://www.360docs.net/doc/066463194.html,

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noma-associated fibroblasts(CAFs;see the Supple-fold(p=0.02)when compared to those of tumors co-mental Experimental Procedures,Isolation of Human

mingled with corresponding counterpart fibroblasts or Breast Fibroblasts and Cell Culture,in the Supplemen-normal fibroblasts,respectively(Table1).

tal Data available with this article online).We also iso-

We also confirmed that the initially injected GFP-lated from each of the same six patients a second pop-labeled MCF-7-ras cells,when comingled with CAF1 ulation of fibroblasts,taken from a noncancerous

cells,contributed prominently to the resulting recon-region of the breast at least2cm from the outer tumor structed tumors by demonstrating strong immunoreac-margin.We termed these cells“counterpart fibro-

tivity against GFP in essentially all cancer cells within blasts.”All experiments were performed by comparing the tumors(Figures1Ca and1Cb).This observation ex-these pairs of CAFs and corresponding counterpart fi-

cludes the possibility that contaminating breast cancer broblasts,thereby avoiding bias due to interindividual cells,originally present in the tumors from which the differences.In addition,we extracted fibroblasts from

fibroblast populations were prepared,were responsible a sample obtained from a reduction mammoplasty,in for growth of the observed tumors.In addition,the co-which only normal mammary tissue was detectable.

inoculated CAF1cells(Figure1Cc),as well as the coun-We then verified the purity of the various fibroblast terpart and normal fibroblasts(data not shown),survived populations by immunostaining.These fibroblast pop-

in large numbers in tumors together with carcinoma ulations strongly expressed fibroblastic markers such cells for periods of up to9weeks after injection,as as vimentin(Figures1Aa and1Ab),prolyl4-hydroxylase

determined by immunohistochemistry using an anti-(Figures1Ad and1Ae),fibronectin,and fibroblast sur-body specific for human vimentin,which MCF-7-ras face protein(data not shown),whereas these cells were

cells fail to express.

negative for cytokeratin(Figures1Ag and1Ah).We also These initial experiments were extended using pairs found that no more than0.1%of the cells in each fibro-

of CAF and counterpart fibroblast populations from five blast population were positive for CD31,CD45,CD3e,other patients,using,as before,admixed normal fibro-CD11b,CD45R/B220,Ly6G,Ly-6C,and TER-119using

blast populations as controls.We observed that four flow cytometry(data not shown).Taken together,these out of six CAF populations were more competent in en-observations indicate that these fibroblast populations

hancing MCF-7-ras tumor growth than were the pa-were prepared with minimal contamination by epithe-tient-specific counterpart fibroblasts,or the normal fi-lial,endothelial,or hematopoietic cells,such as leuko-

broblasts populations(Table1).These observations cytes and erythrocytes.echo findings of others demonstrating that stromal fi-

broblasts isolated from human prostate carcinomas

have an increased ability to foster tumor formation CAFs Are More Competent in Enhancing

when compared to normal prostatic fibroblasts(Olumi Tumor Growth

et al.,1999).

In order to assess the contribution of various fibroblast

We wished to assess the stability of the tumor-populations to tumor growth in vivo,we developed a

enhancing phenotype of the CAFs.To do so,we pas-human tumor xenograft model that enabled us to spec-

saged CAF1cells for up to10population doublings ify the majority of cells constituting both the stromal

(PDs)as pure cultures and then coinjected these cells and epithelial compartments in the engrafted tumors.

with tumor cells into nude mice.These CAFs largely Thus,we mixed CAFs,counterpart fibroblasts,or nor-

retained the tumor-enhancing phenotype,indicating mal fibroblasts with MCF-7-ras human breast cancer

that these cells can maintain this trait in the absence cells in a3:1ratio and inoculated these mixtures subcu-

of ongoing contact with carcinoma cells(Figure1D). taneously in immunodeficient nude mice.The MCF-7-

ras cell line carries an activated ras oncogene allele,

allowing these cells to form tumors in the absence of Cultured CAFs Express Traits of Activated exogenous estrogen stimulation(Kasid et al.,1985).We

Fibroblasts(Myofibroblasts)

also introduced the GFP reporter gene into these cells Expression ofα-smooth muscle actin(α-SMA)is a de-to allow their detection in vivo.

fining characteristic of myofibroblasts(Serini and Gab-In an initial set of experiments,MCF-7-ras cells co-biani,1999).Using anti-α-SMA antibody,we observed mixed with CAFs(CAF1cells in Table1)generated tu-

that the human mammary carcinomas from which we mors of greater volume(Figure1Ba)and weight(Figure had extracted CAFs carried large numbers of myofi-1Bb)than did MCF-7-ras cells that had been mixed

broblasts in their stroma(Figure S1).An increased pro-with other types of fibroblasts prior to injection into portion ofα-SMA-positive myofibroblasts also existed host animals.The tumors that developed in the pres-

in three isolated CAF populations(Figures2Ac and2B) ence of CAF1cells also contained far more GFP-when compared to their counterpart fibroblasts(Fig-labeled carcinoma cells when compared to tumors

ures2Ab and2B)and to normal fibroblasts(Figures2Aa developing in the absence of CAF1cells(Figure1Bc),and2B).This increasedα-SMA expression was largely indicating increased tumor cell proliferation.To further

maintained in the initially characterized CAF1cells for quantify tumor growth rate,we determined the averaged up to nine PDs in vitro(Figure2B),indicating that iso-slope of the tumor growth curves for each tumor type

lated CAFs contain a high proportion of myofibroblasts. using mixed effects model estimation(Pinheiro and Myofibroblasts also exhibit an increased ability to in-Bates,2000;Supplemental Experimental Procedures,

duce collagen gel contraction upon growth in such gels Data Analysis).This statistical estimation indicated that(Hinz et al.,2001).Indeed,we observed that CAF1cells the growth rate of tumors(increase of tumor volume/

contracted collagen gels to a much greater extent than day)admixed with CAF1cells increased by2.8-or1.6-did their corresponding counterpart fibroblasts and two

Fibroblast-Secreted SDF-1Enhances Tumor Growth

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Figure1.Enhanced Tumor Growth Kinetics of MCF-7-ras Breast Cancer Cells Comingled with CAFs

(A)Fibroblastic properties of extracted primary human fibroblasts.CAFs(b,e,and h);Counterpart fibroblasts(a,d,and g);MCF-7-ras cells (c,f,and i)Sections were immunostained by anti-vimentin(a,b,and c),anti-prolyl4-hydroxylase(d,e,and f),or anti-cytokeratin antibodies (g,h,and i).Scale bar,50?m.

(B)MCF-7-ras cells were injected alone or coinjected with various fibroblasts subcutanously into nude mice.Tumor volume(a)was ploted in indicated days.Tumor weight(b)and the number of GFP-labeled MCF-ras cells(c)in each tumor were evaluataed at62days after injection. Error bars depict the standard error around the mean.*p<0.05by Student’s t test.

(C)Sections of GFP-labeled MCF-7-ras tumors comingled with CAFs at60days after injection were immunostained using an anti-GFP antibody(b)or stained by hematoxylin and eosin(H&E)(a).A different section(c)from a CAF-containing tumor was also immunostained using both anti-GFP(pink)and human-specific anti-vimentin(brown)antibodies.Scale bar,100?m.

(D)MCF-7-ras cells were coinjected with CAF1(5or10PDs),counterpart(5PDs),normal fibroblasts(5PDs),or no fibroblasts subcutaneously into nude mice.Error bars depict the standard error of the mean.

independent normal fibroblasts(Figure2C).Interest-tions of activated fibroblasts also possess proportion-

ately greater tumor-enhancing ability.

ingly,this increased contractile ability was observed

in all six independently isolated CAFs when compared

to corresponding counterpart fibroblasts(data not CAFs Enhance Tumor Angiogenesis

shown).CAF1cells passaged for10.5PDs in vitro as Initial cursory examinations of tumor sections contain-pure cultures also contracted collagen gels to nearly ing various types of admixed fibroblasts suggested that the same extent as CAF1cells passaged for five PDs CAFs show an increased ability to stimulate tumor an-(Figure2C).Taken together,these results confirmed giogenesis.We assessed formation of tumor-associ-that the CAFs possessed the properties of myofi-ated microvasculature at55–58days after injection by broblasts and maintained these traits without the con-examining serial sections from xenograft tumors ad-tinued presence of carcinoma cells.Moreover,we mixed with various fibroblasts.Both Masson’s tri-found a statistically positive correlation(p<0.001)be-chrome stain(Figure3Aa)and anti-CD31immuno-tween the contractile abilities of the various CAF pop-staining(Figure3Ad)revealed extensive vascular ulations and their tumor-enhancing powers(Figure S2;formation in tumors containing CAF1cells.In contrast, Supplemental Experimental Procedures,Data Analy-capillaries in tumors containing counterpart fibroblasts sis).Hence,CAF populations having greater propor-

(Figures3Ab and3Ae),normal fibroblasts(Figures3Ac

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Table1.Summary of the Tumor-Promoting Ability in Six Different CAF Populations

CAFs Versus Normal Fibroblasts Versus Counterpart Fibroblasts

Increased

Increased Tumor Increased Increased

Tumor Growth Tumor Tumor Tumor Weight(g)Weight Tumor Weight(g)Rate Weight Tumor Weight(g)Growth Rate CAF10.384(n=10)+++*0.176(n=12)++*++*0.234(n=12)+++* (Std=0.145)(Std=0.054)(p=0.02)(Std=0.083)(p=0.02)

(62days)

CAF20.344(n=13)++*0.207(n=12)++*++*0.225(n=15)++* (Std=0.105)(Std=0.076)(p<0.01)(Std=0.070)(p<0.01)

(68days)

CAF30.381(n=13)++*0.235(n=15)+++*+*0.288(n=12)+* (Std=0.082)(Std=0.110)(p<0.01)(Std=0.079)(p<0.01)

(77days)

CAF40.443(n=15)?0.455(n=13)??0.528(n=11)?(Std=0.184)(Std=0.195)(p=0.7)(Std=0.188)(p=0.7)

(98days)

CAF50.337(n=11)+0.306(n=14)+?0.448(n=11)?(Std=0.144)(Std=0.112)(p=0.8)(Std=0.176)(p=0.8)

(103days)

CAF60.486(n=15)++*0.315(n=15)++*+0.445(n=13)+* (Std=0.143)(Std=0.147)(p<0.01)(Std=0.136)(p<0.01)

(83days)

CAFs and patient-matched,control counterpart fibroblasts were extracted from breast tissues obtained from six human breast cancer patients.In addition,normal breast fibroblasts were isolated from a healthy patient.Either set of described three types of fibroblasts was coinjected with MCF-7-ras cells subcutaneously into nude mice.Weight(g)and growth rate(increase of tumor volume/day)of tumors comingled CAFs were compared to those of tumors containing corresponding counterpart fibroblasts or normal fibroblasts.Thus,relative increased ratios of the averaged weight and growth rate in tumors containing CAFs are represented using an arbitrary scale.Averaged tumor weight(g)measured at the indicated day,the standard deviations of the tumor weights,and the numbers of examined tumors are also shown. *p<0.05by Student’s t test for tumor weight and by mixed effects models for tumor growth rate.Arbitrary scale;+++,more than2-fold up in CAF-containing tumors;++,1.5-to w2-fold up;+,1-to w1.5-fold up;?,not increased;Std,standard deviation.

and3Af),or no fibroblasts(data not shown)were far gle-cell suspensions at60–63days after subcutaneous less developed.Our assessment of the averaged

injection and then used flow cytometry to quantify the microvascular density per tumor type indicated a4.7-Sca1+cell population representing hematopoietic pro-fold greater level in CAF-containing tumors(Figure3Ba)

genitor cells and the Sca1+CD31+EPC population out when compared with tumors containing admixed coun-of the total viable cells in tumors.We found a far higher terpart fibroblasts.Moreover,Chalkley point counting

percentage of Sca1+cells(2.9-fold)and Sca1+CD31+ (numbers of grids covered on each vessel)indicated a EPCs(4.2-fold)in tumors containing CAF1cells when

compared to tumors containing counterpart fibroblasts 7.6-fold greater degree of vascularization in the same

tumors compared to tumors carrying admixed counter-(Figure3Cb).

part fibroblasts(Figure3Bb).Taken together,these ob-

Such EPCs likely arrive in tumors via the circulation, servations indicated that in the presence of CAFs,tu-having been mobilized in the bone marrow and there-mors became more vascularized,suggesting that CAFs

after introduced into the peripheral circulation(Heissig enhance tumor growth,at least in part,by promoting et al.,2002).To examine this possibility,we evaluated tumor neovascularization.

the extent of EPC mobilization into the peripheral blood

of mice harboring either CAF1-,counterpart fibroblast-,

or normal fibroblast-containing tumors.We observed a CAFs Induce Mobilization and Recruitment

higher level of Sca1+(2.1-fold)and Sca1+CD31+(2.0-of Endothelial Progenitor Cells

fold)mononuclear cells in the circulation of mice bear-Tumor angiogenesis has previously been demonstrated

ing CAF1-containing tumors compared to those circu-to occur in part through the recruitment of circulating

lating in mice bearing counterpart fibroblast-containing EPCs(Bertolini et al.,2003;Lyden et al.,2001).We

tumors(Figure3Cc).Moreover,we failed to observe a therefore wished to determine whether the ability of

significant difference in ratios of circulating EPCs in CAFs to promote tumor angiogenesis could be attrib-

mice bearing tumors developing in the absence of uted to an increased ability to recruit EPCs into the tu-

CAFs compared to those in non-tumor-bearing mice. mor masses.In these experiments,we considered

These observations confirmed that Sca1+CD31+cell Sca1+CD31+cells to represent the EPC-enriched cell

populations were indeed mobilized into the circulation fraction(Asahara et al.,1997;Rafii and Lyden,2003);

by the CAF-containing MCF-7-ras tumors in vivo. these cells also showed a20-fold higher ratio of VE-

To address whether these circulating cells could in cadherin-positive cells(an additional specific marker of

fact contribute to angiogenesis,we prepared Sca1+ endothelial cell lineages)when compared to Sca1?

CD31+EPCs or Sca1?CD31?cell fraction from the bone CD31?cells(Figure3Ca).

We dissociated the whole tumor xenografts into sin-marrow of RAG-1-deficient mice(having an H2k-b ma-

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Figure2.CAFs Exhibit Characteristics of“Myofibroblasts”

(A)CAF1cells(c and d),cognate counterpart(b),or normal fibroblasts(a)were cultured in DMEM with2%FCS.Immunostaining with an anti-α-SMA antibody(a,b,and c)or control IgG(d).Scale bar,50?m.

(B)α-SMA-positive cell counts as a fraction of total cell numbers(>100counted cells)were evaluated in nine independent fields from three different wells of each fibroblast type under a fluorescence microscope.*p<0.05.

(C)The area of the contracted gels was measured at the indicated times.The appearance of the contracted gels at72hr is also shown. Error bars depict the standard error of the mean.

jor histocompatibility haplotype)by flow cytometry.three sets of CAFs and the respective patient-specific Subsequently,we injected5×105of these bone mar-

counterpart fibroblasts.These analyses showed an ele-row cells intravenously into haplotype mismatched vation of SDF-1expression in CAFs(data not shown). NOD-SCID(nonobese diabetic,severe combined im-

We therefore quantified expression levels of both munodeficient)mice(of an H2k-d haplotype)bearing SDF-1a and b mRNAs,which are alternative splicing 20-day-old MCF-7-ras tumors.As shown in Figure3Cd,

forms of a common pre-mRNA,in the various stromal at21days after injection of either bone marrow-derived fibroblast https://www.360docs.net/doc/066463194.html,ing real time RT-PCR,We cell preparation,we found that many tumor-associated

found increased mRNA levels of both SDF-1a(Figure capillaries(revealed by their display of CD31antigen)4Aa)and b(Figure4Ab)in CAF1(2.8-and 5.0-fold,

respectively),CAF2(2.3-and2.0-fold),and CAF3(5.6-were composed largely of cells staining positively for

the H2k-b haplotype(Figure3Cd3)by immunofluores-and2.3-fold)cell populations when compared to the cence.This demonstrates that circulating Sca1+CD31+

cognate counterpart fibroblasts of each set of CAFs. EPCs were,in fact,capable of differentiating into vas-In addition,we performed an ELISA assay on the me-cular endothelial cells in tumor-associated capillaries.

dium conditioned by each CAF population using an an-In contrast,in mice in which control Sca1?CD31?cells tibody that reacts with both SDF-1αandβforms.This had been injected intravenously,the tumor-associated

assay indicated elevated levels of SDF-1protein in the vasculature failed to incorporate cells of the H2k-b medium conditioned by the CAF1(1.8-fold),CAF2(2.6-

fold),and CAF3(3.1-fold)cells when compared to the haplotype(Figure3Cd4).These observations indicated

that one of the tumor-enhancing functions of CAFs de-levels produced by the respective counterpart fibro-rives from their ability to mobilize EPCs and to recruit

blast populations(Figure4Ac).We failed to observe them into the tumor mass.Once localized within tu-higher levels of SDF-1in CAF4and CAF5,CAFs that mors,such EPCs are capable of differentiating into tu-

had previously not shown any tumor-enhancing ability mor-associated vascular endothelial cells.in vivo(data not shown).Finally,MCF-7-ras cells

showed a far lower level of SDF-1secretion when com-CAFs Express High Levels of Stromal Cell-Derived

pared to CAFs(Figure4Ac).

Factor-1(SDF-1)We then performed immunostaining using both anti-Several secreted factors,such as vascular endothelial

SDF-1and anti-α-SMA antibodies on sections of inva-growth factor(VEGF),stromal cell-derived factor-1sive breast carcinomas obtained from human patients. (SDF-1),soluble c-Kit ligand,and matrix metalloprotei-

We observed that the subset of fibroblast-like cells nase9(MMP9),have been implicated as possible regu-positive forα-SMA(as indicated by arrows in Figure lators of EPC proliferation,mobilization,and migration

4Bb)also were positive for SDF-1(as indicated by ar-(Heissig et al.,2002).In order to examine factors in-rows,Figure4Ba)in tumor stroma.Notably,theα-SMA

protein present on myofibroblasts in tumor stroma was volved in the mobilization and recruitment of EPCs,we

performed DNA microarray expression analysis on largely colocalized with the SDF-1protein(Figure4Bf).

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Figure3.MCF-7-ras Tumors Developing in the Presence of CAFs are Highly Angiogenic and Recruit Increased Numbers of EPCs

(A)Sections from MCF-7-ras tumors containing various fibroblasts were stained by anti-CD31antibody(d,e,and f)or by Masson’s trichrome (a,b,and c).Scale bar,100?m.

(B)Increased microvascular density(a)and Chalkley counts(b)in tumors comingled with CAFs**p<0.01.Error bars depict the standard error of the mean.

(C)(a)Mouse bone marrow cells were stained by anti-Sca1,-CD31,and-VE-cadherin antibodies.(b)MCF-7-ras tumors developing in the presence of various fibroblasts were dissociated into single cells at60–63days after injection.(c)Peripheral blood cells were also harvested from mice,and the positive cells out of the total viable cells were quantified using anti-Sca1and anti-CD31antibodies.*p<0.05.(d)Frozen sections were generated from tumors in mice injected intravenously with Sca1+CD31+EPCs(1,2,and3)or control Sca1?CD31?bone marrow cells(4).The sections were double-immunostained using both anti-H2k-b(1)and anti-CD31(2)antibodies and double-positive cells are shown in a merged view(3).Scale bar,50?m.

In contrast,we failed to detect any fibroblast-like cells To this end,we developed an in vitro transwell chemo-

taxis assay.GFP-labeled bone marrow fractions were positive for SDF-1in noncancer stroma(as shown by

arrowheads,Figure4Bc).As previously reported(Pab-sorted by flow cytometry to enrich for EPCs using anti-los et al.,1999),we detected the SDF-1protein to some

Sca1and anti-CD31antibodies,as before.Either Sca1+ extent in normal epithelial cells(as indicated by arrows,CD31+EPCs or control Sca?CD31?bone marrow cells Figure4Bc),breast carcinoma cells(depicted by an ar-

were then seeded into the upper wells of Boyden rowhead,Figures4Bd and4Bf)and endothelial cells chambers,and the lower wells of these chambers were

seeded with confluent layers of various stromal fibro-(data not shown).Together,these findings indicate that

the myofibroblasts present within breast carcinomas blast populations.

produce higher levels of SDF-1in vivo than do stromal

GFP-positive cells that migrated into the lower well fibroblasts present in noncancer stroma.were counted under a microscope18hr after initial co-

culturing.We found a3.3-fold increase in the chemo-SDF-1Released by CAFs Mediates the Chemotaxis

taxis index of the Sca1+CD31+EPCs when cocultured and Recruitment of EPCs with CAF1cells in the lower well(Figure5A)when com-We wished to test whether SDF-1released from CAFs

pared with cognate counterpart fibroblasts.Importantly, plays a functional role in stimulating EPC recruitment.treatment with an anti-SDF-1neutralizing antibody(10or

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341

Figure4.CAFs Produce Increased SDF-1at Both mRNA and Protein Levels

(A)SDF-1a(a)and b(b)mRNA levels in various fibroblasts were quantified by real time PCR.SDF-1protein concentration(c)in the medium conditioned by these cells was measured by ELISA.Error bars represent the standard errors of the mean in three independent experiments. *p<0.05

(B)Invasive human breast carcinoma sections were immunostained with anti-SDF-1(a,c,d,and f)and anti-α-SMA(b,e,and f)antibodies. SDF-1+α-SMA+myofibroblasts are shown in a merged view(f).Scale bar,75?m.

20?g/ml)inhibited the increased chemotaxis toward8.6-fold higher in the Sca1+CD31+fraction when com-CAFs in a dose-dependent manner,whereas a control

pared to the Sca1?CD31?fraction(Figure5B),explain-mouse IgG failed to show this effect(Figure5A).More-ing the far higher SDF-1-dependent migration of the over,chemotaxis toward counterpart or normal fibro-

Sca1+CD31+fraction.

blasts was not affected by the anti-SDF-1neutralizing As an additional control,we verified that the migrated antibody(Figure5A).In contrast,the Sca1?CD31?frac-

Sca1+CD31+cells in these chamber assays were in-tion of the bone marrow cells showed a lower degree deed EPCs by assessing their colony-forming ability of chemotaxis that was largely SDF-1-independent and

upon long-term culture.After our transwell chemotaxis unaffected by the type of fibroblasts cultured in the migration assay,the mixed cell populations in the lower

wells were cultured in the absence of the upper wells lower well(Figure5A).Furthermore,the percentage of

cells expressing CXCR4—the receptor for SDF-1—was for an additional5weeks.CAF-containing cultures de-

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Figure5.SDF-1Mediates the Increased Migration of EPC-Enriched Cells toward CAFs

(A)In vitro transwell chemotaxis assays were performed using four to five independent wells of each fibroblast type.The chemotaxis index was calculated as the ratio of the number of either Scal+CD31+EPCs or Scal?CD31?cells that migrated toward a particular fibroblast population to the number of these cells that migrated toward medium-only wells.**p<0.01.GFP-positive EPCs migrated on CAFs(d), counterpart(c)-,normal(b)-or nonfibroblast(a)layers.Scale bar,100?m.

(B)Bone marrow cells were stained with anti-CXCR4,anti-Sca1,and anti-CD31antibodies.Error bars represent the standard error around the mean in three independent experiments.

(C)Colonies formed on CAF1cells were evaluated by FITC-conjugated Ulex europaeus agglutinin I(lectin)binding(a)and DiI-labeled acet-ylated-LDL uptake(b).Merged images are also shown(c).Scale bar,50?m.

veloped EPC colonies(Figure5Cc)that were double-Petit et al.,2002)into nude mice bearing MCF-7-ras positive for DiI-labeled acetylated-LDL uptake and tumors admixed with either CAF1cells or counterpart FITC-conjugated Ulex europaeus agglutinin I(lectin)fibroblasts.Mice received intraperitoneal injections of binding,both of which are markers of EPCs(Kalka et antibodies twice a week for a period of68days follow-al.,2000).These observations,when taken together,in-ing the initial injection of cells.

dicate that CAFs have an enhanced ability in recruiting Significantly,the resulting MCF-7-ras tumors con-EPCs via SDF-1release in vitro.taining CAFs in mice treated with an anti-SDF-1anti-

body showed greatly attenuated tumor growth(Figure

6Aa),reduced tumor volume,and decreased tumor SDF-1Released by CAFs Contributes

weight(Figure6Ab)compared to those of CAF-contain-to the Recruitment of EPCs,

ing tumors in mice treated with control IgG.The CAF-Angiogenesis,and Tumor Growth

containing tumors in mice treated with an anti-SDF-1 We next examined whether SDF-1released by CAFs

antibody also exhibited a53%reduction in microvascu-did indeed play an essential role in enhancing tumor

lar density(Figure6Ac).Moreover,the proportion of growth and angiogenesis through EPC recruitment into

Sca1+CD31+EPCs out of the total viable cells in the the tumors.To do so,we administrated an anti-SDF-1

neutralizing antibody(Coulomb-L’Hermin et al.,1999;tumors was decreased by36%compared to CAF-con-

Fibroblast-Secreted SDF-1Enhances Tumor Growth

343

Figure6.An Anti-SDF-1Neutralizing Antibody Attenuated the Increased Growth of MCF-7-ras Tumor Developing in the Presence of CAFs, Decreased Angiogenesis,and Reduced Recruitment of EPCs into Tumors

(A)Mice bearing MCF-7-ras tumors comixed with CAF1cells or control counterpart fibroblasts were intraperitoneally treated with either anti-SDF-1antibody or control mouse IgG twice a week for a period of68days after initial injection of the tumor cells.We examined tumor growth kinetics(a),tumor weight(b),angiogenesis(c)and the percentage of Sca1+CD31+EPCs(d)within the tumor masses.*p<0.05.

(B)(a)Recombinant SDF-1αprotein was added to cultured MCF-7-ras cells in the presence of either control IgG or anti-CXCR4neutralizing antibody(20?g/ml).Total numbers of MCF-7-ras cells in each of four independent wells were counted at days3and5.(b)Two independent CXCR4-siRNA or control GFP-siRNA lentivirus vectors were introduced into MCF-7-ras cells.CXCR4protein expressed on the surface of MCF-7-ras cells was then analyzed using an anti-CXCR4antibody by flow cytometry.(c)MCF-7-ras cells expressing various siRNAs or non-siRNA were cultured in the presence or absence of SDF-1(100ng/ml).Total cell numbers in each of four independent wells were counted at day5.

Error bars depict the standard error of the mean.

taining tumors treated with control IgG(Figure6Ad),CAFs in vivo plays a crucial role in enhancing tumor whereas the anti-SDF-1antibody failed to affect signifi-

growth and angiogenesis by recruiting EPCs into tu-cantly any of the above parameters in those tumors mor masses.

formed by MCF-7-ras cells to which control counterpart

fibroblasts had been admixed(Figure6A).Stromal SDF-1Enhances Tumor Growth Significantly,as gauged by an ELISA assay,cultured

by Paracrine Stimulation of Tumor Cells

CAFs secreted SDF-1at a far higher level(33-fold Recent reports have indicated that SDF-1boosts the greater in the case of CAF1cells)than did MCF-7-ras

proliferation of several cancer cell lines in culture,in-cells,as shown in Figure4Ac.These findings argue that cluding breast carcinoma cells(Allinen et al.,2004;Hall

and Korach,2003).For this reason,we tested whether the inhibitory effect of the anti-SDF-1neutralizing anti-

body used in our xenograft model acted largely by in-CAF-secreted SDF-1could also directly stimulate MCF-hibiting SDF-1secreted by stromal CAFs,rather than

7-ras cell proliferation.Thus,we first treated MCF-7-by inhibiting SDF-1released by the MCF-7-ras cancer ras cells with recombinant SDF-1αprotein in culture. cells.These observations,together with those de-

Indeed,SDF-1(applied at1,10,or100ng/ml)stim-scribed earlier,indicate that the SDF-1released by ulated MCF-7-ras cell proliferation in a dose-dependent

Cell

344

Figure7.Schematic Representation of Tumor-Promoting Effects Provoked by Stromal Fibroblasts within Invasive Human Mammary Carci-nomas

(A)MCF-7-ras tumor cells expressing either control GFP-siRNA,CXCR4-siRNA-1,or-2vectors were admixed to SDF-1α-or control GFP-expressing normal human breast fibroblasts.These mixtures were then injected subcutaneously into nude mice.In each group,statistically significant difference of tumor volume is indicated by a bucket line.*p<0.05.Error bars depict the standard error of the mean.

(B)Two mechanisms by which SDF-1released by stromal fibroblasts in invasive human mammary carcinomas facilitates tumorigenesis. Stromal fibroblast-derived SDF-1enhances tumor growth both by stimulating angiogenesis through recruiting circulating EPCs into the tumor mass(endocrine effect)and by direct paracrine stimulation of tumor cells through CXCR4expressed on carcinoma cells(paracrine effect).

manner,generating1.2-,1.37-or1.5-fold increases in We found that MCF-7-ras tumors expressing either cell numbers,respectively,when compared to non-

CXCR4-siRNA vector showed more attenuated tumor SDF-1-treated MCF-7-ras cells(Figure6Ba).Moreover,growth kinetics and significantly reduced tumor volume an anti-CXCR4neutralizing antibody(20?g/ml),which

(p<0.05)in the presence of SDF-1α-expressing fibro-should block the ability of the CXCR4receptor to bind blasts compared to MCF-7-ras tumors expressing a

control GFP-siRNA vector(Figure7A).This observation its SDF-1ligand,largely inhibited the SDF-1-induced

proliferation(Figure6Ba),implicating CXCR4displayed demonstrates that stromal SDF-1enhances tumor by the MCF-7-ras cells as the mediator of this prolifera-

growth in vivo in part by direct paracrine stimulation tion effect.through CXCR4on MCF-7-ras breast cancer cells.

We then determined whether direct paracrine stimu-

We also found that MCF-7-ras tumors carrying either lation of tumor cells by stromal SDF-1occurs in vivo.CXCR4-siRNA vector admixed with control GFP-Furthermore,we sought to confirm that SDF-1acts

expressing fibroblasts grew more slowly and had a sig-through the CXCR4on the tumor cells themselves in nificantly decreased tumor volume(p<0.05)than did addition to other CXCR4-expressing cells such as

tumors arising from these same carcinoma cells that EPCs.To do so,we constructed MCF-7-ras cells un-had been commingled with SDF-1α-expressing fibro-able to respond to SDF-1by introducing lentiviral vec-

blasts(Figure7A).This result shows that stromal SDF-1,as tors expressing CXCR4-siRNA into these cells.The re-proposed above,also boosts tumorigenesis by affect-sulting significant inhibition of CXCR4receptor protein

ing other CXCR4-expressing cells besides the MCF-7-expression(by73%–78%)in the MCF-7-ras cells by ras tumor cells;these other targets might include the

EPCs studied in the earlier experiments.Together, either of two CXCR4-siRNA lentivirus vectors(Figure

6Bb)completely abrogated the ability of SDF-1to stim-these findings strongly suggest that CAF-secreted ulate MCF-7-ras cancer cell proliferation(Figure6Bc).

SDF-1promotes carcinoma growth in vivo by direct In contrast,a control GFP-siRNA construct failed to paracrine stimulation through CXCR4present on breast have this effect.Importantly,the loss of CXCR4did not

carcinoma cells in addition to its endocrine effects that affect MCF-7-ras cell proliferation in the absence of allow EPC recruitment into tumors(Figures7A and7B). SDF-1(Figure6Bc),indicating that the CXCR4-siRNA

vectors did not retard MCF-7-ras cell proliferation Discussion

through nonspecific effects.

In addition,we generated normal human mammary While the presence of fibroblasts in the stroma of inva-

sive mammary carcinomas is well documented,the stromal fibroblasts expressing retroviral SDF-1α-or

control GFP-expression vectors.SDF-1α-expressing specific contributions of these fibroblasts to tumor fibroblasts exhibited an18-fold higher level of SDF-1

growth have been unclear.We have shown here that protein,as measured by ELISA,when compared to fibroblasts present in invasive human mammary carci-control GFP-expressing fibroblasts(Figure S3).We ad-

noma masses are biologically very different from their mixed MCF-7-ras cells carrying CXCR4-or GFP-siRNAs counterparts located outside of the tumor masses in

several important functional respects.(1)Fibroblasts with SDF-1α-or control GFP-expressing fibroblasts

before injecting them subcutaneously into nude mice.extracted from within invasive human breast cancer

Fibroblast-Secreted SDF-1Enhances Tumor Growth

345

masses are more competent than their counterparts in Our work takes these other observations still further, enhancing tumor growth by comingled breast cancer by demonstrating that carcinoma cells can utilize the cells.(2)These fibroblasts exhibit increasedα-SMA ex-normal host stromal response by converting recruited pression as well as increased contractility,both indica-cells to CAFs,which in turn recruit EPCs into the tumor tive of myofibroblasts.(3)When comingled with breast via SDF-1(Figure7B).While CAFs contribute in a major cancer cells,these fibroblasts give rise to highly vascu-way to the recruitment of EPCs,it remains to be seen larized tumors.(4)These fibroblasts produce increased whether CAF-derived SDF-1is also involved in recruit-levels of SDF-1.(5)The SDF-1released by these fibro-ing leukocytes into tumors.Fibroblast-secreted SDF-1, blasts is responsible for recruiting EPCs into tumor for example,plays a key role in maintaining chronic in-masses,thereby boosting tumor angiogenesis.In addi-flammation by regulating the mobilization,recruitment, tion,the SDF-1produced by these CAFs enhances tu-and immigration of leukocytes into inflammatory tis-mor growth by direct paracrine stimulation via CXCR4sues(Douglas et al.,2002;Suratt et al.,2004).More-displayed by human breast carcinoma cells.over,chronic inflammation aids carcinoma growth by Significantly,the tumor-enhancing capability of CAFs generating a tumor-prone microenvironment(Coussens is largely maintained,at least during10PDs in vitro,in and Werb,2002).Furthermore,the recruitment of the absence of continuous interaction with carcinoma CD11b+Gr-1+leukocytes contributes to tumor angio-cells.The present experiments do not address how genesis by producing angiogenic factors,such as CAFs acquire these unique phenotypes and how these MMP9,and by directly differentiating into endothelial cells maintain the myofibroblastic phenotypes.Thus,cells(Yang et al.,2004).These lines of evidence support during tumor formation,preexisting mammary stromal the possibility that CAF-secreted SDF-1assists tumor fibroblasts or recruited progenitors(Ishii et al.,2003)progression in part through an inflammatory response. may be induced by nearby carcinoma cells to convert Importantly,endogenous CXCR4expression on car-into the myofibroblasts.Alternatively,myofibroblasts or cinoma cells is known to correlate with a poor progno-their progenitors may be recruited from outside of the sis for several types of carcinomas(Balkwill,2004; tumor mass.Staller et al.,2003),although relatively little is known Stromal regions microdissected from human breast about the role of its ligand,SDF-1,in primary tumor cancers show a high frequency of genetic alterations,growth.It is also known that CXCR4ectopically ex-such as loss of heterozygosity(LOH)and somatic mu-pressed on carcinoma cells enhances primary tumor tations(Fukino et al.,2004;Kurose et al.,2002;Moinfar growth in a mouse xenograft model(Darash-Yahana et et al.,2000).These observations raise the possibility al.,2004)and that the knockdown of CXCR4expression that an accumulation of genetic alterations may con-in breast carcinoma cells abrogates the tumor growth tribute to the CAFs’activated,tumor-enhancing pheno-(Lapteva et al.,2005;Smith et al.,2004).Moreover,a types.We note,however,that our CAFs show no detecta-small-molecule inhibitor of CXCR4reduces primary ble aneuploidy as determined by karyotype analysis,no brain tumor growth(Rubin et al.,2003).Thus,it is likely anchorage-independent growth in culture,and no tu-that SDF-1secreted by stromal myofibroblasts signifi-morigenicity in vivo(data not shown).Moreover,some cantly affects CXCR4-expressing human breast carci-of our CAFs begin to senesce after15PDs in culture.nomas through direct paracrine stimulation.Inhibition It is also quite likely that CAFs carry epigenetic alter-of the activated SDF-1/CXCR4signal pathway in a pri-ations,such as the generation of an autocrine TGF-βmary tumor environment may therefore attenuate carci-loop that can convert normal fibroblasts into myofi-noma growth in breast cancer patients.

broblasts(Ronnov-Jessen et al.,1996;Serini and Gab-

biani,1999).Importantly,CAFs exhibit activated pheno-

Experimental Procedures

types(e.g.,increased contractility)very similar to those

of myofibroblasts within wound sites.Such non-carci-Immunostaining of the Human Fibroblasts

noma-derived myofibroblasts may also have the poten-and Breast Cancer Tissues

tial to aid tumor growth.If such capability is indeed

For double immunostaining of vimentin and GFP in paraffin sec-

tions of MCF-7-ras tumors,the DAKO Envision Doublestain System observed,this would indicate that CAFs,such as those

was used.Primary cultured fibroblasts or frozen sections from hu-characterized here,are not unique to the stroma of car-

man breast carcinomas were also examined by immunofluores-cinomas and that once myofibroblasts are formed,

cence using the antibodies indicated below.

such cells may already be fully competent to exert tu-

mor-enhancing effects.

Antibodies

We have demonstrated that SDF-1secreted from

Primary antibodies used for staining included anti-pan-cytokeratin CAFs recruits EPCs into carcinomas,enhancing angio-(Sigma),human specific anti-vimentin(V9;Novocastra Laborato-genesis and thus tumor growth.This observation mir-ries,Ltd.,United Kingdom),anti-fibronectin(Sigma),anti-prolyl rors the observation that the tumor stroma resembles

4-hydroxylase(5B5;Dako,Denmark),anti-fibroblast surface pro-

tein(1B10;Sigma),anti-human CD31(Dako,Denmark;Santa Cruz, that present in sites of tissue injury(Dvorak,1986).

California),α-SMA(1A4;Dako,Denmark),FITC-conjugated anti-Thus,SDF-1released from injured tissues recruits

H2k-b,phycoerythrin(PE)-conjugated anti-CD31(BD PharMingen), EPCs into injured sites,inducing tissue regeneration

anti-SDF-1(K15C),and anti-GFP(Abcam,United Kingdom). (Askari et al.,2003;Ceradini et al.,2004;De Falco et

al.,2004;Schober et al.,2003).Moreover,our finding

Measurement of Collagen Gel Contraction

that CAFs release higher levels of SDF-1is supported

by Human Breast Fibroblasts

by recent work indicating that myofibroblasts extracted2×105fibroblasts were suspended in300?l of collagen lattices from human mammary tumors express increased levels prepared in24-well plates,and4–5independent collagen gels in-of the SDF-1mRNA(Allinen et al.,2004).

cluding various fibroblasts were cultured in DMEM with2%FCS.

Cell

346

The assay was performed as previously described(Bogatkevich et munostained by FITC-conjugated anti-H2k-b and PE-conjugated al.,2001).

anti-CD31antibodies.

Subcutaneous Tumorigenicity Assays Flow Cytometry

One million MCF-7-ras cells and three million fibroblasts of various Extracted human fibroblasts were stained by biotin-conjugated types were coinjected subcutaneously into nude mice.Tumorige-CD45,CD3e,CD11b,CD45R/B220,Ly6G,Ly-6C,and TER-119anti-nicity assays were performed as previously described(Elenbaas et bodies(BD PharMingen).Streptavidin(SA)-allophycocyanin(APC) al.,2001).was then used for detection of biotin-positive cells.106mononu-

clear cells from mouse peripheral blood or bone marrow,106cul-

tured MCF-7-ras cells,or106cells dissociated from xenograft tu-Plasmid Construction

mors were incubated for20min at4°C with the following The GFP gene was cloned into the pWZL-Blasticidin retroviral vec-

antibodies:Anti-FITC-or PE-conjugated Sca1,anti-PE-or APC-tor.The human SDF-1a cDNA(Shirozu et al.,1995)was also cloned

conjugated CD31,VE-cadherin,anti-FITC-conjugated mouse CXCR4, into the pBabe-puro retroviral vector.For inhibition of endogenous

or anti-PE-conjugated human CXCR4(12G5)(BD PharMingen). CXCR4expression in MCF-7-ras cells,oligonucleotides against hu-

Cells incubated with7-amino-actinomycin D(7-AAD;BD Phar-man CXCR4or GFP genes were generated and cloned into a lenti-

Mingen)were analyzed using flow cytometry(Becton Dickinson). virus-derived pLKO-puro-RNAi vector(Stewart et al.,2003).The fol-

lowing oligomers were used:CXCR4-siRNA-1,5#-TGGAGGGGAT

CAGTATATACA-3#;CXCR4-siRNA-2,5#-GTTTTCACTCCAGCTAAC In Vitro Transmigration Assay

ACA-3#as previously described(Anderson et al.,2003;Doench et1×105fibroblasts were seeded in the lower well of a24-well cham-al.,2003);GFP-siRNA,5#-GCAAGCTGACCCTGAAGTTCA-3#.ber two days prior to coculture.GFP-labeled Sca1+CD31+or

Sca1?CD31?cell populations were sorted from whole bone marrow

cells from GFP-transgenic mice(Ikawa et al.,1995)using flow cyto-Retroviral and Lentiviral Infections

metry.4×104of these bone marrow cells were then seeded in Retrovirus infection was performed as previously described(Elen-

DMEM+2%FCS in the upper well of a transwell chamber with a baas et al.,2001).pBabe-puro-SDF-1a or-GFP vectors were used

5?m pore size(Costar,Massachusetts).Cells were incubated with to infect normal human breast tissue-derived fibroblasts immortal-

either anti-SDF-1neutralizing antibody or control IgG one day prior ized by hTERT,the catalytic subunit of telomerase.A pWZL-Blas-

to coculture.

ticidin-GFP vector was also introduced by infection into MCF-7-

ras cells.Lentivirus infection of either siRNA-CXCR4or siRNA-GFP

vector was performed as previously described(Stewart et al.,MCF-7-ras Cell Proliferation Assay in Culture

2003).After infection,MCF-7-ras cells were selected by2?g/ml1×104MCF-7-ras cells were cultured in DMEM with2%FCS in puromycin for3.5days and used for experiments on the following the presence or absence of SDF-1α(1–100ng/ml).An anti-CXCR4 day.neutralizing antibody(12G5;20?g/ml)or control mouse IgG was

added12hr prior to treatment with SDF-1α.4×104MCF-7-ras Real Time PCR cells carrying different siRNAs were also cultured in the presence Quantitative real time RT-PCR analysis was performed as pre-or absence of SDF-1α(100ng/ml).Total cell numbers of MCF-7-viously described(Brisken et al.,2002).Data were normalized rela-ras cells in each of four independent wells in24-well plates were tive to the expression level ofβ2-microglobin for each sample.counted at the indicated days.

Primers used for RT-PCR were human SDF-1a,5#-TGAGAGCTC

GCTTTGAGTGA-3#(sense)and5#-CACCAGGACCTTCTGTGGAT-3#Treatment with Anti-SDF-1Neutralizing Antibody(K15C) (antisense);SDF-1b,5#-CTAGTCAAGTGCGTCCACGA-3#(sense)in Mice Bearing Tumors

and5#-GGACACACCACAGCACAAAC-3#(antisense);and b2-micro-MCF-7-ras cells admixed with either CAFs or control counterpart globin,5#-TGAGTGCTGTCTCCATGTTTGA-3#(sense)and5#-TCTG fibroblasts were injected subcutaneously into nude mice.K15C(32 CTCCCCACCTCTAAGTTG-3#(antisense).?g/mouse)or control isotype IgG2(32?g/mouse)antibodies were

intraperitoneally injected starting one day prior to cell injection and Immunoassay for Human SDF-1subsequently injected twice weekly at the same dose(32?g/ 1.5×106fibroblasts were cultured in DMEM with2%FCS on a10mouse)for a period of68days.

cm culture plate for2days.The supernatants were measured using

a commercially available SDF-1ELISA kit(R&D Systems).

Supplemental Data

Evaluation of Angiogenesis in Xenograft MCF-7-ras Tumors Supplemental Data include three figures and Supplemental Experi-Serial sections(taken at2mm intervals)were prepared from MCF-mental Procedures and can be found with this article online at 7-ras tumors(comingled with various fibroblasts or no fibroblasts).https://www.360docs.net/doc/066463194.html,/cgi/content/full/121/3/335/DC1/.

A total of30sections from six independent tumors of each group

were then immunostained using an anti-CD31antibody.Microves-

sel density was assessed as described previously(Weidner et al.,Acknowledgments

1991).After taking these counts,a49-point Chalkley point eyepiece

graticule(Klarmann Rulings,Inc.,New Hampshire)was employed We would like to thank Dr.Todd R.Golub for help of DNA microar-over the same tissue section and the number of graticular points ray experiment and critical reading of this manuscript,Drs.Ittai (at×400magnification)that fell within the lumen of vessels was Ben-Porath,Kimberly Hartwell,Christina Scheel,Nir Hacohen, counted(Fox et al.,1995).Konrad Hochedlinger,and Chengcheng Zhang for critical reading

of this manuscript and Drs.Paul Matsudaira,S.A.Mani,and Sridhar Colony Formation Assay of Endothelial Progenitor Cells Ramaswamy for helpful experimental suggestions and data analy-After5weeks of coculture in DMEM+2%FCS,uptake of DiI-sis and members of R.A.W.’s laboratory for helpful comments and labeled acetylated-LDL and staining of FITC-conjugated Ulex euro-discussion.We appreciate Dr.Charlotte Kuperwasser’s assistance paeus agglutinin I(lectin)were examined according to a method in the isolation of fibroblasts from human breast tissues,Dr.Tasuku previously described(Hatzopoulos et al.,1998).Honjo for gift of the human SDF-1cDNA,and Dr.Ittai Ben-Porath

for a pLKO1-GFP-siRNA vector.This work was supported by Incorporation of EPCs into MCF-7-ras Xenograft Tumors

Merck/MIT(R.A.W.),NIH/NCI grant R21CA87081-02(R.A.W.),the NOD-SCID(H2k-d haplotype+)and RAG-1-deficient mice(H2k-b Ludwig Trust(R.A.W.),the Breast Cancer Research Foundation haplotype+;Jackson Laboratory,)were used.Sca1+CD31+or

(R.A.W.),Uehara Memorial Foundation(A.O.),Sankyo Foundation Sca1?CD31?bone marrow fractions from the RAG-1-deficient mice of Life Science(A.O.),and a US Army Pre-doctoral Breast Cancer were intravenously injected into the NOD-SCID mice bearing MCF-

Fellowship DAMD17-02-1-0468(P.B.G).R.A.W.is an American Can-7-ras tumors.Frozen sections from the resulting tumors were im-cer Society Research Professor and a Daniel K.Ludwig Cancer

Fibroblast-Secreted SDF-1Enhances Tumor Growth

347

Research Professor.This work was conducted utilizing the W.M.Coussens,L.M.,and Werb,Z.(2002).Inflammation and cancer.Na-Keck Foundation Biological imaging facility at the Whitehead In-

ture420,860–867.

stitute.Cunha,G.R.,Hayward,S.W.,Wang,Y.Z.,and Ricke,W.A.(2003).

Role of the stromal microenvironment in carcinogenesis of the

prostate.Int.J.Cancer107,1–10.

Received:February26,2004

Revised:November30,2004Darash-Yahana,M.,Pikarsky,E.,Abramovitch,R.,Zeira,E.,Pal,B., Accepted:February25,2005Karplus,R.,Beider,K.,Avniel,S.,Kasem,S.,Galun,E.,and Peled, Published:May5,2005 A.(2004).Role of high expression levels of CXCR4in tumor growth,

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