The Macrophage Growth Factor CSF1 in Mammary Gland Development and Tumor Progression

P1: GDX

Journal of Mammary Gland Biology and Neoplasia (JMGBN) pp587-jmgbn-378820 September 19, 2002 12:26 Style file version June 22, 2002

Journal of Mammary Gland Biology and Neoplasia, Vol. 7, No. 2, April 2002 ( C© 2002)

The Macrophage Growth Factor CSF-1 in Mammary GlandDevelopment and Tumor Progression

Elaine Y. Lin,1 Valerie Gouon-Evans,1 Andrew V. Nguyen,1,2 and Jeffrey W. Pollard1,3

Colony stimulating factor 1 (CSF-1), a major regulator of the mononuclear phagocytic lin-eage, is expressed in more than 70% of human breast cancers and its expression is correlatedwith poor prognosis. Studies of CSF-1 null mutant mice demonstrated that CSF-1 plays animportant role in normal mammary ductal development as well as in mammary tumor pro-gression to metastasis. CSF-1 regulates these processes through the recruitment and regulationof macrophages, cells that become associated with mammary tumors and the terminal end budsat the end of the growing ducts. This phenomenon suggests that the tumors subvert normaldevelopmental processes to allow invasion into the surrounding stroma, a process that givesthe tumor access to the vasculature and consequently the promotion of metastasis. In addition,soluble CSF-1 secreted from the tumor acts to divert antitumor macrophage responses andsuppresses the differentiation of mature tumor-antigen-presenting dendritic cells. This reviewdiscusses these observations in detail and attempts to fit them into a larger picture of CSF-1and macrophage action in the regulation of normal mammary gland development and tumorprogression.

KEY WORDS: CSF-1; macrophages; mammary; malignant; osteopetrotic.

INTRODUCTION

In mice, mammary gland development is initi-ated at the end of the rudimentary ducts by the for-mation of complex multilaminate, highly prolifera-tive terminal end buds (TEB) (1). These TEBs growout through the fat pad and by bifurcation lay downthe branching pattern. They recruit a unique stromaconsisting of fibroblasts (1,2) and, as we have re-

1 Center for Study of Reproductive Biology and Women’s Health,and the Departments of Developmental and Molecular Biologyand Obstetrics and Gynecology and Women’s Health, AlbertEinstein College of Medicine, New York.

2 Present address: Department of Microbiology and Immunology,Albert Einstein College of Medicine, 1300 Morris Park Avenue,New York, New York 10461.

3 To whom correspondence should be addressed at Center for Studyof Reproductive Biology and Women’s Health, and the Depart-ments of Developmental and Molecular Biology and Obstetricsand Gynecology and Women’s Health, Albert Einstein collegeof Medicine, Chanin Building 607, 1300 Morris Park Avenue,New York, New York 10461; e-mail: [email protected].

cently shown, the hematopoietic cells, eosinophils,and macrophages (3). In a similar fashion, breast tu-mors also surround themselves with a unique stroma

Abbreviations used: APC, antigen-presenting cells; CM, con-ditioned medium; CSF-1/M-CSF, colony stimulating factor1/macrophage-colony stimulating factor; Csf1op or op, os-teopetrotic mutation; CSF-1R, CSF-1 receptor; DC, dendriticcells; ImDC, immature dendritic cells; MDC, mature dendriticcells; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3, EGF, epidermalgrowth factor; GFs, growth factors; HIFs, hypoxia-induced tran-scription factors; IL-4, interleukin-4; IL-6, interleukin-6; iNOS,inducible nitric oxide synthase; MCP-1, monocyte chemoattrac-tant protein-1; mCSF-1, membrane associated isoform of CSF-1;MØ, macrophage; MMP, matrix metalloproteinase; MMTV LTR,the mouse mammary tumor virus long terminal repeat; NO,nitric oxide; NOS, nitric oxide synthase; PA, plasminogen ac-tivator system; PAI-1, plasminogen activator inhibitor type 1;PyMT, polyoma middle T antigen; PD-ECGF/TP, thymidinephosphorylase/platelet-derived endothelial cell growth factor;RANK, receptor activator of nuclear factor κB; RCC, renal cellcarcinoma; TAM, tumor-associated macrophage; TEB, terminalend buds; Tg, transgene; TGF, transforming growth factor; TP,thymidine phosphorylase; uPA, urokinase-type plasminogen ac-tivator; VEGF, vascular endothelial growth factor.

1471083-3021/02/0400-0147/0 C© 2002 Plenum Publishing Corporation

P1: GDX


148 Lin, Gouon-Evans, Nguyen, and Pollard

whose composition can modulate tumor progressioneither negatively or positively (4). This tumor stromaenvironment is also rich in hematopoietic cells, withflorid leukocytic infiltrations often being evident (5).Macrophages are a major component of these leuko-cytic infiltrations (6). The roles of these macrophagesin tumors have been a subject of much debate andcontroversy. One view suggests that these cells areinvolved in tumor rejection through their immuno-logical function, while the opposing view is that theypromote tumor growth and invasion (7). In this reviewwe will present evidence that these macrophages aretrophic to tumor cells in a manner similar to their rolein normal mammary development.

Colony stimulating factor-1 (CSF-1), also knownas Macrophage (M)-CSF, is the major regulator of themononuclear phagocytic lineage [for review see (8)].Substantial evidence exists in a variety of human tu-mors, particularly those of the female reproductivesystem, including breast cancer, that poor progno-sis is correlated with overexpressions of CSF-1 (9).These findings led to our hypothesis that CSF-1, to-gether with other cytokines/chemokines, regulatesmacrophage recruitment to the developing tumorin a similar fashion to the developing mammarygland (10). In both these locales the macrophagesprovide trophic factors that allow the outgrowth ofthe cells through the stroma. In the case of the tumor,however, mutations in epithelial cells lead to the lossof positional identity, such that they are able to invadeinto the blood stream and metastasize to distant sites.This review will discuss the data that support this hy-pothesis and will suggest ways of tilting the balanceaway from trophic roles of macrophages in tumors tothose functions that could result in tumor rejection.

CSF-1 AND ITS RECEPTOR

The CSF-1 gene is localized to human chromo-some 1p13-p21 (11) and to mouse chromosome 3 atthe osteopetrotic locus (12). The human and mousegenes comprise 10 exons and nine introns (13). Sev-eral human and murine cDNAs have been clonedfrom mature alternative transcripts (mRNA), rangingin size from 1.6 to 4.0 kb (8,13). The heterogeneityof CSF-1 mRNAs results from differential splicing ofcoding exon 6 and of the 3′ noncoding region (ex-ons 9 and 10) of the gene together with differentialpolyadenylation site selection (14). These mRNAs en-code different membrane-spanning precursor formsthat yield either the membrane-spanning cell sur-face glycoprotein, the secreted glycoprotein, or the

proteoglycan forms of the growth factor. The lattertwo are released with a rapid half-life (∼23 min) whilethe cell surface form can be clipped from the surfacewith a t1/2 of approximately 11 h. All of these solu-ble and cell surface forms are biologically active ashomodimers (Fig. 1)[for review see (8,14)].

The effects of CSF-1 are mediated by a highaffinity cell surface transmembrane receptor tyrosinekinase (CSF-1R) that is encoded by the c-fms pro-tooncogene product (15). The CSF-1R belongs to theclass 3 family of tyrosine kinase receptors that in-cludes platelet-derived growth factor receptors α andβ, and the c-kit protooncogene product. It has a highlyglycosylated extracellular domain characteristic ofmembers of the immunoglobulin gene superfamilyand a tyrosine kinase domain that is interrupted by aninterkinase domain of 73 amino acids (16). Binding ofthe homodimeric ligand results in receptor dimeriza-tion, auto-trans tyrosine phosphorylation, and propa-gation of the signal to a variety of downstream kinasesubstrates as illustrated in Fig. 1. Extensive studies us-ing cells expressing either the murine or human CSF-1receptor in fibroblasts demonstrated that kinase ac-tivity and various receptor tyrosine phosphorylationsites are required for CSF-1R signaling. These stud-ies, together with those on primary macrophages andmacrophage cell lines, have shown that CSF-1 signal-ing can result in cell survival, proliferation, differen-tiation, and movement [reviewed in (17)].

BIOLOGICAL ACTIVITY OF CSF-1

Initial studies with cells of the hematopoietic or-gans and cell lines of other cell types indicated thatthe CSF-1R was restricted to mononuclear phago-cytic cells, from the determined but undifferentiatedmonoblast through to the mature macrophages. Usingbone-marrow-derived macrophages and early bonemarrow progenitors, it was established that CSF-1 isrequired for survival, differentiation, and prolifera-tion of cells of the mononuclear phagocytic lineage(18,19). In addition, CSF-1 is chemoattractive andchemokinetic for macrophages (20). CSF-1 has beenshown to be produced by a wide variety of cells in cul-ture, either constitutively at low levels or after stim-ulation (21). However, the fact that CSF-1 is part ofthe early response gene repertoire (22) suggests thatmany of these cells produce CSF-1 as a result of thestress of culture conditions. Consistent with this idea,in vivo expression of CSF-1 is more restricted bothtemporally and spatially and is only found at specificlocations in a wide range of tissues, including the

P1: GDX


Fig. 1. CSF-1 genomic organization, transcripts, proteins, and interaction with CSF-1R. The top half of the diagram depictsthe expression of CSF-1 including the intron–exon relationships and cDNA clones encoding different forms of CSF-1 protein.Exons (1–10), 5′ and 3′ untranslated regions (line) and coding region (box), including signal peptide (∗) and transmembranedomain (hatched), are indicated, together with the N-linked (filled arrowheads) and O-linked (open circle) glycosylationsites and the site of glycosaminoglycan addition (open diamond). Filled regions of CSF-1 homodimers are those presentin the mature secreted or released glycoprotein forms while both filled and opened regions are present in the majorsecreted proteoglycan form. The glycosaminoglycan chain is shown as linked, open circles. The hatched region representsthe transmembrane domain. The lower half of the diagram represents the ligand-induced changes in CSF-1R prior tointernalization of the receptor–ligand complex. The boxed regions indicate some of the proteins tyrosine phosphorylatedas a result of CSF-1R activation that transduce the action of CSF-1 on mononuclear phagocyte survival, differentiation,proliferation, and motility [modified from (14) and (8)].

149

P1: GDX



mammary epithelium (23). In these locations, localsynthesis of CSF-1 probably serves to recruit and reg-ulate particular populations of mononuclear phago-cytes. An example of this behavior is the expression ofCSF-1 in osteoblasts to localize and regulate receptor-bearing osteoclasts at sites of bone remodeling (24).CSF-1 is also a serum growth factor, probably de-rived from endothelial cells, in which it serves to reg-ulate circulating monocyte populations [for reviewsee (25)].

The understanding of CSF-1 biology was greatlyenhanced by the identification of the naturally occur-ring osteopetrotic mutation, Csf1op, formerly op, asan insertion mutation in the CSF-1 gene that resultsin a frame shift within the mRNA and prematuretermination of translation (14,26). Consequently,homozygous mutants express only a truncated,nondimerized, biologically inactive form of CSF-1.This complete lack of CSF-1 results in a depletion ofmany populations of mononuclear phagocytes (27)and osteoclasts, whose absence results in the char-acteristic osteopetrosis, after which the mutation isnamed (14). Studies on these mice confirmed thecentral role in vivo for CSF-1 in the regulation ofthe mononuclear phagocytic lineage (28). However,detailed studies revealed populations of mononuclearphagocytes whose presence is independent of CSF-1(27–29). These phagocytes tend to be associated morewith immune function and include dendritic cells(27,30), while those whose tissue location is CSF-1-dependent are more often associated with scavengingand trophic functions (Fig. 2) (14). It should benoted however, that the function of the mononuclearphagocytes whose presence is independent of CSF-1could still be functionally dependent on CSF-1,since they express the CSF-1R (14). Indeed, ourrecent studies with Listeria monocytogenes-infectedCsf1op/Csf1op mice revealed that CSF-1-regulatedmacrophage functions were essential for adequateimmune defenses (31). Recently, a null mutation wasintroduced into the CSF-1 receptor (CSF-1R) gene,and mice homozygous for this mutation showedalmost identical phenotypes to the ligand null (32).This study provides genetic evidence that the c-fmsgene product is the only receptor for CSF-1.

The absence of CSF-1 signaling in mice resultsin several phenotypic consequences other than os-teopetrosis, including toothlessness, dermal hyper-plasia, impaired neuronal processing, and signif-icant reproductive deficiencies in both male andfemale mice (14,26,28). Consequent analyses of thesemice have led to a greater appreciation of the role

of macrophages in many diverse processes beyondthe conventional roles in immunity [reviewed in(14,33,34)], as well as a unique role for CSF-1 actingthrough receptor-bearing trophoblastic cells in pla-cental immune regulation (35). A recent report hasshown that CSF-1-promoter-driven CSF-1 transgeneexpression in Csf1op/Csf1op mice completely rescuedtheir diverse phenotypes together with all the popu-lations of macrophages (23), confirming the role ofCSF-1 as the major, but not the only, regulator ofmacrophages in vivo.

CSF-1 IN BREAST CANCER

Overexpression of CSF-1 CorrelatesWith Poor Prognosis

Multiple early studies have documented abnor-mal expression of CSF-1/CSF-1R in a wide variety ofhuman carcinoma-derived cell lines, including tumorsof epithelial origin such as those of breast, ovary, andendometrium (36–39). Clinical studies have shownthat in breast cancer CSF-1 expression was found in∼75% of cases, and the extent of expression in thesetumors correlates with high grade and poor progno-sis (40). Circulating CSF-1 has been used as a tumormarker for the detection and monitoring of responseto therapy in patients with ovarian, breast, and en-dometrial carcinoma (40–42). In breast carcinoma,CSF-1 expression is prevalent in invasive tumor cellscompared with intraductal cancer (43). CSF-1 expres-sion is also found in association with expression ofCSF-1R in tumor cells (∼50% of all tumors) and infil-trated macrophages (90% of tumors) suggesting bothautocrine and paracrine roles for CSF-1 in tumor in-vasion (43). In patients with breast cancer the over-expression of both CSF-1 and its receptor is stronglycorrelated with poor prognosis (43,44) and is predic-tive of recurrence for ipsilateral breast cancer (45). Ina recent prospective study of over 600 breast cancerpatients followed for 67 months, CSF-1 levels weremarkedly elevated in those patients with aggressivelocally advanced or metastatic tumors (46). Together,these clinical correlative studies suggest that CSF-1has a causal role in the progression of breast cancerthrough both autocrine and paracrine effects.

Autocrine Effects of CSF-1 in Breast Cancer

Evidence supporting the hypothesis that CSF-1has a direct effect on mammary tumor cells comes

P1: GDX


Macrophages, Mammary Gland Development, and Cancer 151

Fig. 2. CSF-1 regulation of cells of the mononuclear phagocytic lineage. Shown are the common mononuclear phagocytic precursorthat gives rise to the osteoclastic lineage and population of macrophages that have trophic and scavenging roles, both of which aredependent on CSF-1. In contrast, macrophages that populate the immune organs are relatively independent of CSF-1 in terms of tissuedensity; however, CSF-1R is expressed by these cells and many of their functions require CSF-1; op indicates stages of differentiationthat are affected by the osteopetrotic mutation at the Csf1op locus [modified from (34)].

from both in vitro and in vivo studies. Studies us-ing mammary carcinoma or epithelial cell lines haveshown that CSF-1 regulates their tumorigenicity andinvasiveness. Transfection of a cDNA for the CSF-1Rinto a noninvasive, CSF-1R-negative mouse mam-mary epithelial cell line, HC11, that produces CSF-1resulted in a 100-fold increase in invasiveness as mea-sured by migration through a barrier of reconstitutedbasement membrane and the enhanced ability to formcolonies in soft agar compared with the nontrans-fected cells (47). The opposite effect, of a complete in-hibition of colony formation in soft agar and abolish-ment of the CSF-1-stimulated invasion was achievedusing a dominant negative mutant of Ets-2 in an Ets-2-positive breast cancer carcinoma cell line, BT20 (48).Ets-2 is a transcription factor that plays a centralrole in mediating CSF-1 responses (49). In additionto mammary carcinomas, CSF-1-enhanced invasive-ness was also observed in other epithelial carcinomas.

For example, CSF-1 together with dexamethasone,which increases CSF-1R levels, enhanced the in vitroinvasiveness of CSF-1R-positive lung carcinoma celllines (50). These studies suggest that tumor-producedCSF-1 may act as an autocrine factor promotinggrowth and the invasion of tumor cells into the stroma.

Paracrine Effects of CSF-1 in Breast Cancer

Tumor-produced CSF-1 may also have aparacrine role through its action on macrophages.These cells are the major components of the lym-phoreticular infiltrate of various forms of solid tumorsand can comprise up to 50% of the cell mass in breastcarcinomas (6,51,52). CSF-1 is a potent macrophagechemoattractant factor (20) and may be responsiblefor the recruitment of macrophages to the tumor site.Suggestions that these leukocytes play a role in tumorprogression dates back to Virchow in 1863 and later

P1: GDX



to the concept that tumors are wounds that neverheal (53). However, until recently their functionalsignificance was largely ignored (54). Indeed, studieson the role of macrophages have been rather con-tradictory, with reports of both positive and nega-tive effects of macrophages on tumor growth [(7);see Leek and Harris, 2002, 177–189]. Macrophagesare known to mediate direct antitumor cytotoxicityand to present tumor antigens (55,56). On the otherhand macrophages can also induce immunosuppres-sion against tumor targets (57). In nonimmunologicalcontexts, macrophages can promote tumor angiogen-esis (58,59) in a wide range of situations (60). Hypoxiain tumors recruits macrophages to those sites (61) andinduces hypoxia-induced transcription factors (HIFs)in both macrophages and in tumor cells. HIFs pro-mote the expression of angiogenic factors includingVEGF (62). Consistent with this observation is thepositive correlation between macrophage infiltrationand tumor angiogenesis in breast cancers (63–65). In-deed, using Chalkley count morphometry in invasivebreast carcinomas, Leek et al. demonstrated both apositive correlation between high vascular grade andincreased macrophage index, and a strong relation-ship between increased macrophage counts and re-duced relapse-free survival and overall survival (64).Nevertheless, many of the experimental studies on theroles of macrophages have been performed on cellsin culture or in xenograft situations where host versusgraft responses might be engendered. To clarify therole of CSF-1 and macrophages in mammary tumorsarising within the host environment, we analyzed tu-mor progression in mammary tumors arising as a re-sult of expression of the oncoprotein Polyoma middleT antigen (PyMT) in the mammary epithelium in theabsence and presence of physiological and supraphys-iological concentrations of CSF-1. The resultant datareviewed below strongly support a trophic role formacrophages in tumor progression and metastasis.

CSF-1-REGULATED MACROPHAGESPROMOTE TUMOR PROGRESSIONAND METASTASIS IN THE MOUSE

CSF-1 Accelerates Mammary Tumorsto Metastases in the Mouse

To examine the effects of the absence of CSF-1in tumor progression, we crossed CSF-1 null mutantmice, Csf1op/Csf1op, with the breast cancer susceptiblePyMT mice, in which the expression of the oncogene,Polyoma middle T antigen, was directed into the

mammary epithelium by the mouse mammary tumorvirus long terminal repeat, MMTV LTR (66). Ourstudy showed that mammary tumors occurred in allcontrol wild type+/Csf1op and mutant Csf1op/Csf1op

PyMT mice at an early age as a single tumor focusin the ducts emanating from the nipple, followed byother tumors in ducts distant to the nipple. The growthrate of the primary nonmalignant tumors in controland mutant PyMT mice increased linearly over 4–10weeks. However, at 10 weeks of age a dramatic pro-gression of the primary tumor to malignant stages oc-curred in+/Csf1op PyMT mice. In 50% of these micethe primary tumors had developed to the most malig-nant late carcinoma stage, but only 13% of primary tu-mors in mutant mice at this age had developed to thisstage. Following the progression of the primary tumorto malignancy, pulmonary metastases were detectedin+/Csf1op PyMT mice at 18 weeks of age but not inCsf1op/Csf1op PyMT mice, even when they reached 22weeks of age (10). These results show that the absenceof CSF-1, while not affecting tumorigenesis and thegrowth of the primary tumor, resulted in delayed tu-mor progression and an almost complete abrogationof lung metastases.

To determine whether CSF-1 acts in a local fash-ion in the mammary gland to regulate tumor pro-gression, we developed mouse strains that specificallyrestored secreted CSF-1 to the mammary gland byexpressing CSF-1 under the control of the MMTVpromoter in the mammary epithelium. We madethe expression of this CSF-1 transgene tetracycline-repressible by using a binary system so that we couldcontrol the expression of CSF-1 temporally by ad-ministration of the tetracycline analogue doxycycline.The mouse lines expressing both transgenes MMTV-tTA and tetop-CSF-1 were bred with+/Csf1op PyMTmice. We found that in the CSF-1 transgenic PyMTCsf1op/Csf1op mice both the progression of primarymammary tumor to carcinoma and the incidence ofpulmonary metastases were accelerated to levels sim-ilar to that found in PyMT +/Csf1op littermates,suggesting that CSF-1 regulates mammary tumorprogression in an organ-autonomous manner. Toeliminate the possibility that this phenomenon wasrestricted to mutant mice because of their alteredmammary development (see later), we also overex-pressed CSF-1 in +/Csf1op PyMT control mice thatare phenotypically normal with respect to the serumCSF-1 level, hematopoiesis, and mammary gland de-velopment (14). We found that both the local pro-gression of the tumor in the mammary gland and dis-tant pulmonary metastases in these mice were greatly

P1: GDX



Fig. 3. Tumor metastasis is associated with the infiltration of CSF-1-dependent macrophages in primary mammary tu-mors. Shown are transverse sections of primary mammary tumors from PyMT mice immunostained with monoclonal an-tibody against macrophage lineage-specific marker F4/80. Primary tumors were prepared from +/Csf1op(+/op), CSF-1 null-mutant, Csf1op/Csf1op(op/op) or Csf1op/Csf1op PyMT mice expressing CSF-1 transgene in mammary gland (op/op CSF-1Tg)(magnification ×250). Note that, compared with the tumor from the CSF-1 null-mutant mouse, +/op or op/op CSF-1 Tgtumors, which had normal or elevated level of CSF-1 in mammary glands, had higher density of F4/80-positive cells at thetumor vicinity (open arrow). This increase of F4/80 positive cells at primary tumor site was correlated with a higher incidenceof pulmonary metastasis.

accelerated (10). Thus, local overexpression of CSF-1not only resulted in an enhanced tumor progressionin Csf1op/Csf1op PyMT mice but also accelerated thisprocess in wild type PyMT mice expressing normallevels of systemic CSF-1. These data provide strongsupport for the hypothesis that CSF-1 plays an im-portant role in the transition of tumors from the non-malignant to malignant state and in their ability tometastasize.

Macrophage Infiltration Positively CorrelatesWith Mammary Tumor Progression in Mice

A close association between macrophage den-sity and mammary tumor progression was observedin PyMT mice (10)(Fig. 3). A dramatic increase of

leukocytic infiltration containing many macrophagesoccurred in the primary mammary tumors in+/Csf1op

PyMT mice prior to the transition to malignancy. Nosuch leukocytic infiltration was observed at the tu-mor site in Csf1op/Csf1op PyMT mammary glands,although the primary tumors in both genotypeshad progressed to similar nonmalignant stages (ade-noma). As the primary tumors developed to thecarcinoma stage in +/Csf1op PyMT mice, the infil-tration of leukocytes became more intense. Denselyinfiltrated cells with the morphology of granulocytes,mast cells, and monocyte-like cells were found inthese sites, and the tumor acini adjacent to them of-ten displayed a disrupted boundary. This observationsuggested that the basement membrane of the aciniat the infiltration site had lost its integrity, allowing

P1: GDX



tumor cells to migrate into the adjacent connectivetissue. The density of the infiltrated cells was reducedin Csf1op/Csf1op PyMT tumors, even in those thathad developed to the carcinoma stage (Fig. 3). In-terestingly, in situ hybridization for CSF-1R mRNAexpression showed that the sole localization of theCSF-1R is in macrophages, with no expression in tu-mor cells (10). These results are consistent with thehypothesis that CSF-1 acts through macrophages inits promotion of tumor progression.

To test the hypothesis that CSF-1 acts lo-cally on tumor-associated macrophages (TAM), themacrophage density as assessed by positivity for themononuclear phagocyte restricted antigen, F4/80, inthe MMTV-CSF-1 transgenic PyMT mice describedabove was examined (10). Correlated with the acceler-ation of malignant transition in the CSF-1 transgenicCsf1op/Csf1op PyMT mice was a marked increase offocal infiltration sites of F4/80+ cells in the mammarytumor (Fig. 3). Furthermore, the density of infiltratingleukocytes and F4/80+ cells in the mammary glandsof CSF-1-transgenic +/Csf1op PyMT mice was alsogreater than in control +/Csf1op PyMT mice. In ad-dition, this infiltration occurred much earlier in theCSF-1 transgenic mice compared with the nontrans-genic controls, and this correlated histologically withadvanced tumor grade. Taken together, these datahave identified CSF-1 as an important regulatory fac-tor in mammary tumor progression to metastasis, act-ing through the intermediary of TAMs.

DIFFERENTIAL ROLES FOR CSF-1ISOFORMS IN TUMOR PROGRESSION

CSF-1 may have opposite roles in tumor pro-gression according to its isoform expression. An in-hibitory effect of membrane-associated CSF-1 in tu-mor progression in rat glioma has been reported. Invitro studies showed that T9 glioma cells transfectedwith cDNAs encoding only the membrane isoformof CSF-1 (mCSF-1) were killed by bone-marrow-derived macrophages (67,68). Interestingly, T9 cellstransfected with the secreted isoform of CSF-1were not killed when cocultured with macrophages.The macrophage-mediated killing of the mCSF-1-transfected T9 cells was blocked by excess recombi-nant soluble CSF-1 (68). Evidence obtained in vivoalso supports differential roles for CSF-1 isoforms.Intracranial implantation of mCSF-1-transfected T9glioma cells resulted in an 80% rejection of the tumorand consequent survival of the rat, whereas similar

injections of either the parental T9 or T9 cells express-ing the secreted form of CSF-1 were 100% fatal (69).The tumor site of mCSF-1-transfected cell also had amarked macrophage infiltration, and many of thesewere in physical contact with the tumor cells. Theseresults indicate that the tumor rejection was, at leastin part, macrophage-mediated (69).

A similar effect of the membrane form of CSF-1in the induction of tumor killing was also found in astudy of the breast cancer cell line, MADB106, whichis weakly immunogenic but highly malignant (70).This study showed that in 48-h cytotoxicity assays,mCSF-1-transfected MADB106 cells were physicallyconjugated with macrophages, but were not killedby them. However, the tumor cells were rejectedwhen these transfectants were inoculated subcuta-neously into normal rats. Furthermore, rats that spon-taneously rejected the mCSF-1-transfected tumorsshowed rechallenge resistance to parental MADB106and to another breast cancer cell line, but not toa glioma cell line. These result suggest that breast-cancer-specific immunity was induced by the in-oculation of mCSF-1-expressing MADB106 tumorcells (70).

Taken together, these studies suggest that dif-ferent isoforms of CSF-1 have opposite effects ontumor biology. The membrane isoform may in-duce macrophage cytotoxicity and systemic immu-nity against tumors while the soluble form of CSF-1may inhibit this immunity. Noteworthy in this con-text is that the majority of the CSF-1 expressed inbreast, endometrial, and ovarian tumors is the se-creted form (38–40).

MODULATION OF THE IMMUNE RESPONSEIN BREAST CANCER

Impaired immune responses are often observedin cancer patients or in tumor-bearing animals (71).One mechanism, among a variety of mechanisms thattumor cells have developed to avoid antitumor im-mune responses, is the production of soluble fac-tors that inhibit immune effector functions and im-pair the development of immune cells [for review see(71)]. Dendritic cells (DC) are important antigen-presenting cells (APC) that are members of themononuclear phagocytic lineage that express CSF-1R(72). Several clinical studies have shown that signif-icant defects in responses to tetanus toxoid and in-fluenza virus were found in patients with advanced-stage breast cancer (73). For example, a clinical study

P1: GDX



with a variety of cancers found that the function ofDCs in peripheral blood and tumor draining lymphnodes were equally impaired in these patients, indi-cating a systemic effect of the tumors on DCs (73).This study reported that the number of DCs was dra-matically reduced in the peripheral blood of cancerpatients, an effect closely associated with the stageand duration of the disease (73).

Although the correlation between plasma levelof CSF-1 and accumulation of immature DCs wasnot reported in the above studies, in vitro stud-ies of renal cell carcinoma cell lines (RCC) showedthe importance of CSF-1 in DC differentiation (74).RCC were found to produce soluble factors that in-hibit the differentiation of DC from CD34+ pro-genitors (74). IL-6 and CSF-1 were found to be re-sponsible for the inhibition, since antibodies againstIL-6 and/or CSF-1 abrogated the inhibitory effects ofRCC-conditioned medium (74). Similarly, recombi-nant IL-6 and/or CSF-1 also inhibited the differen-tiation of DC (74). Further studies have shown thatblocking the expression of CSF-1 and IL-6 receptor-transducing chain by treating RCC with IL-4 couldreverse the inhibitory effect of RCC conditioned me-dia on the maturation of DCs (75). Overall, thesestudies indicate that soluble CSF-1 is able to inhibitthe development of immature hemotopoietic cells to-ward DC with antigen-presenting functions. Interest-ingly, these studies have also shown that the RCC re-lease soluble factors that triggered the commitmentof CD34+ progenitors toward monocytic cells with apotent phagocytic capacity (74). Thus a hypothesis isthat tumor-produced CSF-1 together with other cy-tokines recruit TAMs that aid tumor development,while at the same time these factors suppress the de-velopment of antigen-presenting dendritic cells thatmight present tumor-antigens to T cells. This scenariosuggests a possible site of therapeutic interventionthrough the blockage of CSF-1 action.

TROPHIC ROLES FOR TAMs IN THEPROMOTION OF TUMOR INVASIONAND ANGIOGENESIS

Macrophages have many functions that could betrophic to tumors, including matrix remodeling, secre-tion of angiogenic factors and production of growthfactors/cytokines directed toward tumor or stromalcells. One of the systems involving TAMs in the pro-motion of tumor invasion and angiogenesis is the plas-minogen activator system (PA), which is regulatedby CSF-1 [for review see (76)]. The expression of

urokinase-type plasminogen activator (uPA), a serineprotease, as well as plasminogen activator inhibitortype 1 (PAI-1), has been reported to be of clinicaland prognostic value (77). In addition to being in-volved in cancer cell migration and invasion, uPAis known to be involved in tumor angiogenesis andstromal remodeling [for review see (78)]. Analysisof primary breast cancer specimens showed that uPAis localized in both stromal and malignant epithelialcells, whereas the uPA receptor, uPAR, is mainly inspindle- or macrophage-like stromal cells, especiallywhen these cells surround the invasive breast cancer(79). Other studies of breast cancers have shown thatuPAR is found in tumor, stromal, and endothelial cells(80). Studies indicate that TAMs are involved in pro-moting breast tumor progression via the influence oftumor-produced uPA (81). Significant correlations be-tween PAI-1 and vessel remodeling, patient age, nodalstatus, and tumor grade were observed (82). The ex-pression of uPA under the control of CSF-1 had beendemonstrated in macrophages (83,84) and in lung ade-nocarcinomas (85). A recent study, using breast can-cer cell lines well-characterized for differing degreesof invasive, metastatic capability, showed positive cor-relations between expression of CSF-1, level of uPAand tumor invasive ability (86).

Several factors produced by tumor/TAMs havebeen identified to have angiogenic activity in varioustypes of tumors, including thymidine phosphorylase/platelet-derived endothelial cell growth factor (PD-ECGF/TP) [reviewed by (87)], IL-6 (88), VEGF(89), MCP-1 (90), and TGF-beta (91). Activatedmacrophages produce and release nitric oxide (NO)via the enzyme nitric-oxide synthase2 (NOS2) (92).Inducible NOS (iNOS) is expressed predominately instromal cells, including macrophages and endothelialcells of breast and gastric cancers [for review see(92)]. NOS activity can be correlated to cancergrade, suggesting that NO may provide a positivegrowth signal for the tumor (92). In vivo studieshave shown that human tumor cells transfected withiNOS had increased growth rate, vascular density,and invasiveness (92,93). Using a murine breastcancer model, a positive correlation between theexpression of NOS and tumor progression was found,and treatment with NOS inhibitors had antitumorand antimetastatic effects (93).

In addition, macrophages produce many growthfactors, such as epidermal growth factor (EGF),that are chemotactic to tumor cells (94). Conse-quently, these growth-promoting substances mightenhance tumor cell migration from the primary site,

P1: GDX



or stimulate tumor cell proliferation or viability. Allof these effects could influence tumor progression andhelp to select cells that are more aggressive. Taken to-gether, these studies indicate that TAMs can promotetumor growth, invasion, and angiogenesis through avariety of mechanisms.

TUMOR-PRODUCED CSF-1 MAY PROMOTEBONE METASTASIS

Bone is a frequent site of breast cancer metastasis[for review see (95)]. These metastases frequently re-sult in bone destruction (osteolysis) that is mediatedby factors produced or induced by tumor cells throughthe stimulation of osteoclast maturation and activa-tion (95). CSF-1 is a major regulator of the osteoclasticlineage, which is a branch of the mononuclear phago-cytic lineage (Fig. 2). It is a viability factor for thelineage precursors and promotes receptor expressionfor the RANK ligand (RANKL), also known as osteo-clastic growth factor, that causes proliferation and dif-ferentiation of these precursors (96). CSF-1 is also ac-tive on mature osteoclasts, promoting their adhesionto bone surfaces and their activity (97). Several stud-ies have shown that breast cancer cells can produceCSF-1 as well as other factors that enhance osteo-clast formation (95,98). In cocultures of macrophagesisolated from primary breast carcinomas and ratosteoblast-like cells on bone slices in the presenceof 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and hu-man CSF-1, numerous multinucleated cells express-ing osteoclastic markers and capable of extensive la-cunar resorption were formed (99). Moreover, theMDA-MB-231 human breast cancer cell line, whichexpresses CSF-1 constitutively and reliably formsbone metastases in mice, stimulates osteoclast forma-tion when cocultured with the bone marrow stromalcell line UAMS-33 (100). Both CSF-1 and RANKL,whose expression were induced in UAMS-33, wererequired for the stimulation of oestoclast forma-tion (100). Consequently it seems likely that CSF-1secreted by the breast tumor metastases in the bonemicroenvironment, together with other factors, playsa crucial role in promoting tumor osetolysis throughtheir promotion of osteoclast maturation.

CSF-1 REGULATES NORMAL MAMMARYGLAND DEVELOPMENT AND TUMORPROGRESSION THROUGH MACROPHAGES

Apart from these roles in tumor progression,macrophages appear to play a role in normal

mammary development. The first suggestion ofthis was in the failure of branching morphogene-sis in mammary glands of the macrophage-deficientCSF-1 null mutant mice, Csf1op/Csf1op, during preg-nancy (101). During mammary gland development,the Csf1op/Csf1op mice display the same rudimentarybranching tree at an early age (2.5 weeks of age)as wild-type mice, but the onset of TEB formationwas delayed by 1 week and thereafter the numberand the growth rate of the ducts across the fat padwas reduced (3). In addition, branching complexitywas reduced in mature mutant mice compared withthe controls. Thus, in adult CSF-1-deficient mice, themammary gland, although filling the fat pad, is at-rophic and poorly branched. During pregnancy, thisstructure also further fails to undergo proper branch-ing morphogenesis.

The osteopetrotic mouse has a number of phe-notypes that could have a bearing on mammary de-velopment, including a perturbed sex steroid hor-mone feedback in the hypothalamus leading to aber-rant estrous cycling (102). Consequently, to definewhether CSF-1 acts directly in the mammary gland,we specifically restored CSF-1 to the mammary glandby expressing CSF-1 under the control of the MMTVpromoter in the Csf1op/Csf1op background using thetransgenic methods described above. This expressiondid not result in correction of serum CSF-1 level,nor of the gross phenotypes of the null mutant micesuch as osteopetrosis, reduced body weight, or theextended estrous cycle found in Csf1op/Csf1op mice.However, it did result in a significant correction ofductal development in the Csf1op/Csf1op mice (103).Thus, these experiments indicate that the mammarygland defects are organ-autonomous because of thelack of CSF-1.

Hematopoietic Cells are Required for MammaryGland Development

In the developing mammary gland, macrophagesare mainly distributed at the neck of the TEB, andare generally not found associated with ductal struc-tures distal to the TEBs (3). Macrophages are alsofound inside the TEBs where they phagocytose deadepithelial cells that are formed during the process ofTEB outgrowth and luminal remodeling (104). Inter-estingly, eosinophils are also recruited to the TEB andthis requires the chemokine eotaxin synthesized inthe mammary gland (3). To confirm the requirementfor hematopoietic cells in mammary development, we

P1: GDX



depleted hematopoietic precursors by whole-body ir-radiation at day 19 of life, a period before the be-ginning of mammary development when mammaryepithelial stem cells are radiation-resistant (3). Inthese mice there is a dramatic reduction in mam-mary morphogenesis compared with controls. Failureof ductal outgrowth can be rescued following bonemarrow transplantation, which restores leukocytenumber in blood, resulting in a dramatic recruitmentof macrophages and eosinophils to the vicinity ofTEBs (3).

The restoration of mammary gland developmentupon rescue of the leukocyte population in the mam-mary gland provides evidence of the necessity ofleukocytes, and more precisely macrophages, in duc-tal outgrowth (3). In a similar fashion, the delayedmammary gland development in CSF-1 null mutantmice, Csf1op/Csf1op, is also associated with a reduc-tion in macrophages around the developing TEBs.Restoration of circulating CSF-1 from day 2 of life toCsf1op/Csf1op mice restored macrophages around theTEBs and largely rescued the ductal outgrowth. Fur-thermore, in the experiments where CSF-1 expressionis restricted to the mammary epithelium (MMTV-tTA and tetop-CSF-1), the correction of ductal de-velopment was associated with a marked increase ofmacrophage density around the TEBs (103). In allof these experiments, macrophages were identified asthe only cells expressing the CSF-1R in the mammaryglands, as assessed by immunohistochemistry usingan anti-CSF-1R antibody (3). Taken together, theseexperiments provide compelling evidence for the re-quirement of macrophages for branching morphogen-esis in the mammary gland.

CONCLUSION

On the basis of these studies, we propose thatCSF-1 produced by the normal or tumor epitheliumacts as a monocyte chemoattractant whose action re-sults in a local accumulation of macrophages in boththe developing mammary gland and tumor. In devel-oping mammary glands, these processes are well reg-ulated and result in an appropriately branched ductaltree filling the fat pad. In contrast, tumor cell growthis unregulated because of intrinsic changes in the ep-ithelial cells that block the recognition of normal po-sitional identity and thus result in exuberant growthand ultimately metastasis. The tumor therefore hasbecome an outlaw tissue that co-opts normal develop-mental signals to its own purpose. Thus, chemoattrac-

tants, including CSF-1, secreted by the normal mam-mary epithelium and the tumor for the recruitmentof macrophages are likely to be similar. In both situa-tions, once macrophages are in situ they provide sev-eral services to the normal ducts and tumors, includingtheir ability to promote angiogenesis, matrix remodel-ing, and the secretion of growth factors that promotecell proliferation and cell movement and inhibit apop-tosis (Fig. 4). Particularly in mammary tumors, theexcess production of CSF-1 triggers massive tissue re-modeling by the invading leukocytes, degradation ofextracellular matrix and basement membrane, induc-tion of angiogenesis, and cell invasion, all processesthat facilitate tumor metastasis. The other result of therelease of large amounts of soluble CSF-1 by breastcancers is the inhibition of the development of an an-titumor immune reaction. As mentioned above, stud-ies of the membrane isoform of CSF-1 have indicatedthat a macrophage- or T-cell-mediated immune re-action could be induced to destroy tumor cells bear-ing this form of CSF-1. This destruction appears torequire cell-to-cell interactions. The large amount ofsoluble CSF-1 produced by tumor cells may saturateCSF-1R expressed upon macrophages or dendriticcells in the tumor microenvironment such that directcontact through the interaction with the membraneform of CSF-1 between these antigen-presenting cellsand the tumor is blocked. Tumor-produced solubleCSF-1 may also inhibit the differentiation of dendriticcells and therefore further inhibit the induction of aT- or B-cell-mediated immune reaction against thetumor (Fig. 4).

Macrophages are one of the first hematopoieticcells to arrive at sites of inflammation or infectionand act as signaling centers to recruit other inflamma-tory cells, such as neutrophils and mast cells, throughtheir synthesis of chemokines (60). A similar arrayof hematopoietic cells is recruited to tumors that ex-press CSF-1. The importance of these groups of cellshas been highlighted by experiments with an experi-mentally induced squamous cell carcinoma model inmice that showed by bone marrow transplantationthat tumor angiogenesis was enhanced by the produc-tion of matrix metalloproteinase 9 produced by mastcells (105). These inflammatory cells may thereforebe promoting agents for tumorigenesis [for review see(106)]. In fact, there is increasing evidence that per-sistent infections or continuous inflammation is a co-factor in carcinogenesis. Recent estimates have sug-gested that as many as 15% of all human cancers mightbe attributed to infectious agents (107). This observa-tion, together with chronic inflammation associated

P1: GDX



Fig. 4. CSF-1 promotes tumor progression and metastasis in multiple ways. Mammary tumors produce a large amount of soluble CSF-1(yellow triangles). This promotes tumor progression by increasing the invasiveness of CSF-1R-bearing tumor cells and the induction ofmacrophage infiltration to the primary tumor site. In the tumor microenvironment, tumor-associated macrophages (TAMs) may producevarious factors that induce tumor invasion (uPA), tumor angiogenesis (VEGF, uPA, NO and TP), and the breakdown of extracellularmatrix and basement membrane (MMPs). All of these processes facilitate tumor metastasis. Soluble CSF-1 produced from breast cancermetastasis can also promote tumor osteolysis by inducing the maturation and activation of osteoclasts in the bone microenvironment. Insome tumors membrane-associated forms of CSF-1 may also be synthesized. This form of CSF-1 may induce immune reactions, includingmacrophage cytotoxicity and T-cell mediated reactions against the tumor. However, tumor cells may inhibit these host-defense reactionsby producing excess amounts of soluble CSF-1 that saturate the CSF-1R on TAMs, blocking their interactions with tumor cells. In addition,tumor-produced soluble CSF-1 may inhibit the differentiation of dendritic cell precursors (ImDC) that further prevent the induction ofT- or B- cell-mediated specific reactions against the tumor through the activation of mature dendritic cells (mDC).

with tumorigenesis, including that of the bowel, liver,bladder, and breast, suggests that a large percent-age of cancers may be affected by the persistent ac-tions of inflammatory cells (108). Consistent with thisview are recent studies showing that treatment withanti-inflammatory drugs that inhibit cyclooxygenase1 and 2 may reduce the risk of colon cancer by asmuch as 50%. These data together suggest approachesto chemoprevention that attack persistent infectionsacting together with drugs that reduce inflamma-tion. Finally, the action of leukocytes, and particularlymacrophages, may also be tilted away from functionsthat provide trophic support to a tumor, to those that

lead to its rejection. Such a strategy might target CSF-1 synthesis or action through antisense approachesor inhibitors of CSF-1R signal transduction. This ap-proach would inhibit macrophage action and maypromote dendritic cells’ differentiation to cells thatpresent tumor-antigens to the host immune system.Such anti-CSF-1 therapy might be coupled with tumorvaccination in an attempt to induce tumor rejection.

ACKNOWLEDGMENTS

We thank Dr. Rosemary Jagus for critical com-ments upon the manuscript. This work was supported

P1: GDX



by grants from U.S. Army DOD#17-97-1-7153 and theAlbert Einstein Comprehensive Cancer Center P30-CA13330. J.W.P. is the Sheldon and Betty E. FeinbergSenior Faculty Scholar in Cancer Research. E.Y.L.and A.V.N. were recipients of National Research Ser-vice Awards 5-T32-AG00194 and AI-CA09060, re-spectively. V. G.-E. was a recipient of DOD postdoc-toral fellowship.

REFERENCES

1. J. M. Williams and C. W. Daniel (1983). Mammary ductal elon-gation: Differentiation of myoepithelium and basal laminaduring branching morphogenesis. Dev. Biol. 97:274–290.

2. M. C. Neville, D. Medina, J. Monks, and R. C. Hovey (1998).The mammary fat pad. J. Mam. Gland Biol. Neoplasia 3:109–116.

3. V. Gouon-Evans, M. E. Rothenberg, and J. W. Pollard(2000). Postnatal mammary gland development requiresmacrophages and eosinophils. Development 127:2269–2282.

4. M. A. Chrenek, P. Wong, and V. M. Weaver (2001). Tumour–stromal interactions. Integrins and cell adhesions as modu-lators of mammary cell survival and transformation. BreastCancer Res. 3:224–229.

5. G. B. Silberstein (2001). Tumour–stromal interactions. Roleof the stroma in mammary development. Breast Cancer Res.3:218–223.

6. C. O’Sullivan and C. E. Lewis (1994). Tumour-associatedleucocytes: Friends or foes in breast carcinoma. J. Pathol.172:229–235.

7. K. Elgert, D. Alleva, and D. Mullins (1998). Tumor-inducedimmune dysfunction: The macrophage connection. J. Leukoc.Biol. 64:275–290.

8. E. R. Stanley (1994). Colony stimulating factor-1 (macrophagecolony stimulating factor). In A. W. Thomson (eds.), TheCytokine Handbook, Academic Press, San Diego, pp. 387–418.

9. S. M. Scholl, P. Crocker, R. Tang, P. Pouillart, and J. W. Pollard(1993). Is colony stimulating factor-1 a key mediator in breastcancer invasion and metastasis? Mol. Carcinog. 7:207–211.

10. E. Y. Lin, A. V. Nguyen, R. G. Russell, and J. W. Pollard (2001).Colony-stimulating factor 1 promotes progression of mam-mary tumors to malignancy. J. Exp. Med. 193:727–740.

11. S. W. Morris, M. D. Valentine, D. N. Shapiro, J. E. Sublett,L. L. Deaven, J. T. Foust, W. M. Roberts, D. P. Cerretti, andA. T. Look (1991). Reassignment of the human CSF1 gene tochromosome 1p13–p21. Blood 78:2013–2020.

12. S. Gisselbrecht, B. Sola, S. Fichelson, D. Bordereau,P. Tambourin, M. G. Malter, D. Simon, and J. -L. Guenet(1989). The murine M-CSF gene is localized on chromosome3. Blood 73:1742–1746.

13. E. S. Kawasaki and M. B. Ladner (1990). Molecular biology ofmacrophage colony stimulating factor. In T. M. Dexter, J. M.Garland, and N. G. Testa (eds.), Colony Stimulating Factors:Molecular and Cellular Biology, Marcel Dekker, New York,pp. 155–176.

14. J. W. Pollard and E. R. Stanley (1996). Pleiotropic roles forCSF-1 in development defined by the mouse mutation os-teopetrotic (op). Adv. Dev. Biochem. 4:153–193.

15. C. J. Sherr, C. W. Rettenmier, R. Sacca, M. S. Roussel, A. T.Look, and E. R. Stanley (1985). The c-fms proto-oncogeneproduct is related to the receptor for the mononuclear phago-cyte growth factor, CSF-1. Cell 41:665–676.

16. C. J. Sherr (1990). Colony stimulating factor-1 receptor. Blood75:1–12.

17. X. F. Csar, N. J. Wilson, K. A. McMahon, D. C. Marks,T. L. Beecroft, A. C. Ward, G. A. Whitty, V. Kanangasun-darum, and J. A. Hamilton (2001). Proteomic analysis ofmacrophage differentiation. p46/52(Shc) Tyrosine phospho-rylation is required for CSF-1-mediated macrophage differ-entiation. J. Biol. Chem. 276:26211–26217.

18. R. J. Tushinski, I. T. Oliver, L. J. Guilbert, P. W. Tynan, J. R.Warner, and E. R. Stanley (1982). Survival of mononuclearphagocytes depends on a lineage-specific growth factor thatthe differentiated cells selectively destroy. Cell 28:71–81.

19. E. R. Stanley, L. J. Guilbert, R. J. Tushinski, and S. H.Bartelmez (1983). CSF-1 – a mononuclear phagocyte lineage-specific hemopoietic growth factor. J. Cell. Biochem. 21:151–159.

20. S. E. Webb, J. W. Pollard, and G. E. Jones (1996). Direct ob-servation and quantification of macrophage chemoattractionto the growth factor CSF-1. J. Cell Sci. 109:793–803.

21. C. W. Rettenmier and C. J. Sherr (1989). The mononuclearphagocyte colony-stimulating factor (CSF-1, M-CSF). Hema-tol. Oncol. Clin. North Am. 3:479–493.

22. R.-P. Ryseck, H. MacDonald-Bravo, and R. Bravo (1991). Themacrophage-colony stimulating factor gene is a growth factor-inducible immediate early gene in fibroblasts. N. Biol. 3:151–157.

23. G. R. Ryan, X. M. Dai, M. G. Dominguez, W. Tong, F. Chuan,O. Chisholm, R. G. Russell, J. W. Pollard, and E. R. Stanley(2001). Rescue of the colony-stimulating factor 1 (CSF-1)-nullizygous mouse (Csf1op/Csf1op) phenotype with a CSF-1transgene and identification of sites of local CSF-1 synthesis.Blood 98:74–84.

24. R. Felix, W. Hofstetter, A. Wetterwald, M. G. Cecchini, andH. Fleisch (1994). Role of colony-stimulating factor-1 in bonemetabolism. J. Cell. Biochem. 55:340–349.

25. P. Roth and E. R. Stanley (1992). The biology of CSF-1and its receptor. Curr. Top. Microbiol. Immunol. 181:141–147.

26. W. Wiktor-Jedrzejczak, A. Bartocci, A. W. Ferrante Jr.,A. Ahmed-Ansari, K. W. Sell, J. W. Pollard, and E. R.Stanley (1990). Total absence of colony-stimulating factor 1 inthe macrophage-deficient osteopetrotic (op/op) mouse. Proc.Natl. Acad. Sci. U.S.A. 87:4828–4832.

27. M. G. Cecchini, M. G. Dominguez, S. Mocci, A. Wetterwald,R. Felix, H. Fleisch, O. Chisholm, W. Hofstetter, J. W. Pollard,and E. R. Stanley (1994). Role of colony stimulating factor-1 inthe establishment and regulation of tissue macrophages duringpostnatal development of the mouse. Development 120:1357–1372.

28. W. Wiktor-Jedrzejczak and S. Gordon (1996). Cytokine reg-ulation of the macrophage (Mφ) system studied using thecolony stimulating factor-1-deficient op/op mouse. Physiol.Rev. 76:927–947.

29. M. Naito, S. Hayashi, H. Yoshida, S. Nishikawa, L. D. Shultz,and K. Takahashi (1991). Abnormal differentiation of tissuemacrophage populations in “osteopetrosis” (op) mice defec-tive in the production of macrophage colony-stimulating fac-tor. Am. J. Pathol. 139.

P1: GDX



30. M. D. Witmer-Pack, D. A. Hughes, G. Schuler, L. Lawson, A.McWilliam, K. Inaba, R. M. Steinman, and S. Gordon (1993).Identification of macrophages and dendritic cells in the os-teopetrotic (op/op) mouse. J. Cell Sci. 104:1021–1029.

31. I. Guleria and J. W. Pollard (2001). Aberrant macrophage andneutrophil population dynamics and impaired Th1 responseto Listeria monocytogenes in colony-stimulating factor 1- de-ficient mice. Infect. Immun. 69:1795–1807.

32. X. Dai, G. R. Ryan, A. J. Hapel, M. G. Dominguez, R. G.Russell, S. Kapp, V. Sylvestre, and E. R. Stanley (2002). Tar-geted disruption of the mouse CSF-1 receptor gene results inosteopetrosis, mononuclear phagocyte deficiency, increasedprimititive progenitor cell frequencies and reproductive de-fects. Blood 99:111–120.

33. J. W. Pollard (1997). Role of colony-stimulating factor-1 inreproduction and development. Mol. Reprod. Dev. 46:54–61.

34. P. E. Cohen, K. Nishimura, L. Zhu, and J. W. Pollard (1999).Macrophages: Important accessory cells for reproductivefunction. J. Leukoc. Biol. 66:765–772.

35. I. Guleria and J. W. Pollard (2000). The trophoblast is a com-ponent of the innate immune system during pregnancy. Nat.Med. 6:589–593.

36. B. M. Kacinski (1997). CSF-1 and its receptor in breast carci-nomas and neoplasms of the female reproductive tract. Mol.Reprod. Dev. 46:71–74.

37. S. Ramakrishnan, F. J. Xu, S. J. Brandt, J. E. Niedel, R. C.Bast Jr., and E. L. Brown (1989). Constitutive production ofmacrophage colony-stimulating factor by human ovarian andbreast cancer cell lines. J. Clin. Invest. 83:921–926.

38. G. Baiocchi, J. J. Kavanagh, M. Talpaz, J. T. Wharton,J. U. Gutterman, and R. Kurzrock (1991). Expression of themacrophage colony-stimulating factor and its receptor in gy-necologic malignancies. Cancer 67:990–996.

39. H. O. Smith, P. S. Anderson, D. Y. K. Kuo, G. L. Goldberg,C. L. DeVictoria, C. A. Boocock, J. G. Jones, C. D. Runowicz,E. R. Stanley, and J. W. Pollard (1995). The role of colony-stimulating factor 1 and its receptor in the etiopathogenesisof endometrial adenocarcinoma. Clin. Cancer Res. 1:313–325.

40. B. M. Kacinski (1995). CSF-1 and its receptor in ovarian, en-dometrial and breast cancer. Ann. Med. 27:79–85.

41. F. V. Price, S. K. Chambers, J. T. Chambers, M. L. Carcangiu,P. E. Schwartz, E. I. Kohorn, E. R. Stanley, and B. M. Kacinski(1993). Colony-stimulating factor-1 in primary ascites of ovar-ian cancer is a significant predictor of survival. Am. J. Obstet.Gynecol. 168:520–527.

42. A. Hakala, B. M. Kacinski, E. R. Stanley, E. I. Kohorn, U.Puistola, J. Risteli, L. Risteli, C. Thomas, and A. Kauppila(1995). Macrophage colony-stimulating factor 1, a clinicallyuseful tumor marker in endometrial adenocarcinoma: Com-parison with CA 125 and the aminoterminal propeptide oftype III procollagen. Am. J. Obstet. Gynecol. 173:112–119.

43. S. M. Scholl, C. Pallud, F. Beuvon, K. Hacene, E. R. Stanley,L. R. Rohrschneider, R. Tang, P. Pouillart, and R. Lidereau(1994). Anti-colony-stimulating factor-1 antibody staining inprimary breast adenocarcinomas correlates with marked in-flammatory cell infiltrates and prognosis. J. Natl. Cancer Inst.86:120–126.

44. E. Sapi and B. M. Kacinski (1999). The role of CSF-1 in normaland neoplastic breast physiology. Proc. Soc. Exp. Biol. Med.220:1–8.

45. M. G. Maher, E. Sapi, B. Turner, A. Gumbs, P. L. Perrotta,D. Carter, B. M. Kacinski, and B. G. Haffty (1998). Prognostic

significance of colony-stimulating factor receptor expressionin ipsilateral breast cancer recurrence. Clin. Cancer Res.4:1851–1856.

46. R. S. McDermott, L. Deneux, V. Mosseri, J. Vedrenne, K.Clough, A. Fourquet, J. Rodriguez, J. M. Cosset, X. Sastre,P. Beuzeboc, P. Pouillart, and S. M. Scholl (2002). Circulatingmacrophage colony stimulating factor as a marker of tumourprogression. Eur Cytokine Netw. 13:121–127.

47. E. Sapi, M. B. Flick, S. Rodov, M. Gilmore-Hebert, M. Kelley,S. Rockwell, and B. M. Kacinski (1996). Independentregulation of invasion and anchorage-independent growthby different autophosphorylation sites of the macrophagecolony- stimulating factor 1 receptor. Cancer Res. 56:5704–5712.

48. E. Sapi, M. B. Flick, S. Rodov, and B. M. Kacinski(1998). Ets-2 transdominant mutant abolishes anchorage-independent growth and macrophage colony-stimulatingfactor-stimulated invasion by BT20 breast carcinoma cells.Cancer Res. 58:1027–1033.

49. S. J. Langer, D. M. Bortner, M. F. Roussel, C. J. Sherr, andM. C. Ostrowski (1992). Mitogenic signaling by colony-stimulating factor 1 and ras is suppressed by the ets -2 DNA-binding domain and restored by myc overexpression. Mol.Cell. Biol. 12:5355–5362.

50. A. E. Filderman, A. Bruckner, B. M. Kacinski, N. Deng, andH. G. Remold (1992). Macrophage colony-stimulating factor(CSF-1) enhances invasiveness in CSF-1 receptor-positive car-cinoma cell lines. Cancer Res. 53:3661–3666.

51. P. M. Kelly, R. S. Davison, E. Bliss, and J. O. McGee (1988).Macrophages in human breast disease: A quantitative im-munohistochemical study. Br. J. Cancer 57:174–177.

52. R. D. Leek, A. L. Harris, and C. E. Lewis (1994). Cytokinenetworks in solid human tumors: Regulation of angiogenesis.J. Leukoc. Biol. 56:423–435.

53. F. Balkwill and A. Mantovani (2001). Inflammation and can-cer: Back to Virchow? Lancet 357:539–545.

54. A. Mantovani, B. Bottazzi, F. Colotta, S. Sozzani, andL. Ruco (1992). The origin and function of tumor-associatedmacrophages. Immunol. Today 13:265–270.

55. I. L. Bonta and S. Ben-Efraim (1993). Involvement of in-flammatory mediators in macrophage antitumor activity.J. Leukoc. Biol. 54:613–626.

56. R. B. Herberman, H. T. Holden, J. Y. Djeu, T. R. Jerrells,L. Varesio, A. Tagliabue, S. L. White, J. R. Oehler, and J. H.Dean (1979). Macrophages as regulators of immune responsesagainst tumors. Adv. Exp. Med. Biol. 361–379.

57. B. al-Sarireh and O. Eremin (2000). Tumour-associatedmacrophages (TAMS): Disordered function, immune sup-pression and progressive tumour growth. J. R. Coll. Surg.Edinb. 45:1–16.

58. R. S. Kerbel (2000). Tumor angiogenesis: Past, present and thenear future. Carcinogenesis. 21:505–515.

59. J. S. Lewis, R. J. Landers, J. C. Underwood, A. L. Harris, andC. E. Lewis (2000). Expression of vascular endothelial growthfactor by macrophages is up-regulated in poorly vascularizedareas of breast carcinomas. J. Pathol. 192:150–158.

60. M. Lingens (2001). Role of leukocytes and endothelial cells inthe development of angiogenesis in inflammation and woundhealing. Arch. Pathol. Lab. Med. 125.

61. L. Turner, C. Scotton, R. Negus, and F. Balkwill (1999).Hypoxia inhibits macrophage migration. Eur. J. Immunol.29:2280–2287.

P1: GDX



62. H. J. Knowles and A. L. Harris (2001). Hypoxia and tumouri-genesis. Breast Cancer Res. 3:318–322.

63. C. E. Lewis, R. Leek, A. Harris, and J. O. McGee (1995). Cy-tokine regulation of angiogenesis in breast cancer: The roleof tumor-associated macrophages. J. Leukoc. Biol. 57:747–751.

64. R. D. Leek, C. E. Lewis, R. Whitehouse, M. Greenall, J. Clarke,and A. L. Harris (1996). Association of macrophage infiltra-tion with angiogenesis and prognosis in invasive breast carci-noma. Cancer Res. 56:4625–4629.

65. K. Engels, S. B. Fox, R. M. Whitehouse, K. C. Gatter, and A. L.Harris (1997). Up-regulation of thymidine phosphorylase ex-pression is associated with a discrete pattern of angiogenesisin ductal carcinomas in situ of the breast. J. Pathol. 182:414–420.

66. C. T. Guy, R. D. Cardiff, and W. J. Muller (1992). Inductionof mammary tumors by expression of polyomovirus middleT oncogenes: A transgenic mouse mode of a metastatic dis-ease. Mol. Cell. Biol. 12:954–961.

67. M. R. Jadus, C. C. Williams, M. D. Avina, M. Ly, S. Kim,Y. Liu, R. Narasaki, C. A. Lowell, and H. T. Wepsic(1998). Macrophages kill T9 glioma tumor cells bearing themembrane isoform of macrophage colony stimulating fac-tor through a phagocytosis-dependent pathway. J. Immunol.160:361–368.

68. M. R. Jadus, M. C. Irwin, M. R. Irwin, R. D. Horansky,S. Sekhon, K. A. Pepper, D. B. Kohn, and H. T. Wepsic (1996).Macrophages can recognize and kill tumor cells bearing themembrane isoform of macrophage colony-stimulating factor.Blood 87:5232–5241.

69. M. R. Graf, M. R. Jadus, J. C. Hiserodt, H. T. Wepsic, andG. A. Granger (1999). Development of systemic immunityto glioblastoma multiforme using tumor cells genetically en-gineered to express the membrane-associated isoform ofmacrophage colony-stimulating factor. J. Immunol. 163:5544–5551.

70. C. C. Williams, H. Trinh, T. V. Tran, Q. Dan, R. Sanchez,C. Delgado, Y. Chen, B. Sippel, E. W. Jeffes, H. T. Wepsic, andM. R. Jadus (2001). Membrane macrophage colony-stimulating factor on MADB106 breast cancer cells does notactivate cytotoxic macrophages but immunizes rats againstbreast cancer. Mol. Ther. 3:216–224.

71. J. E. Ohm and D. P. Carbone (2001). VEGF as a mediator oftumor-associated immunodeficiency. Immunol. Res. 23:263–272.

72. J. Banchereau, F. Briere, C. Caux, J. Davoust, S. Lebecque,Y. J. Liu, B. Pulendran, and K. Palucka (2000). Immuno-biology of dendritic cells. Annu. Rev. Immunol. 18:767–811.

73. B. Almand, J. R. Resser, B. Lindman, S. Nadaf, J. I. Clark, E.D. Kwon, D. P. Carbone, and D. I. Gabrilovich (2000). Clin-ical significance of defective dendritic cell differentiation incancer. Clin. Cancer Res. 6:1755–1766.

74. C. Menetrier-Caux, G. Montmain, M. C. Dieu, C. Bain, M.C. Favrot, C. Caux, and J. Y. Blay (1998). Inhibition of thedifferentiation of dendritic cells from CD34(+) progenitorsby tumor cells: Role of interleukin-6 and macrophage colony-stimulating factor. Blood 92:4778–4791.

75. C. Menetrier-Caux, M. C. Thomachot, L. Alberti, G.Montmain, and J. Y. Blay (2001). IL-4 prevents the blockadeof dendritic cell differentiation induced by tumor cells. CancerRes. 61:3096–3104.

76. P. A. Andreasen, R. Egelund, and H. H. Petersen (2000). Theplasminogen activation system in tumor growth, invasion, andmetastasis. Cell. Mol. Life Sci. 57:25–40.

77. J. A. Foekens, H. A. Peters, M. P. Look, H. Portengen, M.Schmitt, M. D. Kramer, N. Brunner, F. Janicke, M. E. Meijer-van Gelder, S. C. Henzen-Logmans, W. L. van Putten, andJ. G. Klijn (2000). The urokinase system of plasminogen ac-tivation and prognosis in 2780 breast cancer patients. CancerRes. 60:636–643.

78. S. A. Rabbani and A. P. Mazar (2001). The role of the plas-minogen activation system in angiogenesis and metastasis.Surg. Oncol. Clin. N. Am. 10:393–415.

79. S. Kennedy, M. J. Duffy, C. Duggan, C. Barnes, R. Rafferty, andM. D. Kramer (1998). Semi-quantitation of urokinase plas-minogen activator and its receptor in breast carcinomas byimmunocytochemistry. Br. J. Cancer. 77:1638–1641.

80. R. Hildenbrand, W. Glienke, V. Magdolen, H. Graeff, H. J.Stutte, and M. Schmitt (1998). Urokinase receptor localiza-tion in breast cancer and benign lesions assessed by in situ hy-bridization and immunohistochemistry. Histochem. Cell. Biol.110:27–32.

81. R. Hildenbrand, G. Wolf, B. Bohme, U. Bleyl, and A. Steinborn(1999). Urokinase plasminogen activator receptor (CD87)expression of tumor-associated macrophages in ductal car-cinoma in situ, breast cancer, and resident macrophages ofnormal breast tissue. J. Leukoc. Biol. 66:40–49.

82. S. B. Fox, M. Taylor, J. Grondahl-Hansen, S. Kakolyris,K. C. Gatter, and A. L. Harris (2001). Plasminogen activa-tor inhibitor-1 as a measure of vascular remodelling in breastcancer. J. Pathol. 195:236–243.

83. L. F. Fowles, K. J. Stacey, D. Marks, J. A. Hamilton, and D. A.Hume (2000). Regulation of urokinase plasminogen activatorgene transcription in the RAW264 murine macrophage cellline by macrophage colony-stimulating factor (CSF-1) is de-pendent upon the level of cell-surface receptor. Biochem. J.347(Part 1):313–320.

84. K. J. Stacey, L. F. Fowles, M. S. Colman, M. C. Ostrowski,and D. A. Hume (1995). Regulation of urokinase-type plas-minogen activator gene transcription by macrophage colony-stimulating factor. Mol. Cell. Biol. 15:3430–3441.

85. X. H. Pei, Y. Nakanishi, K. Takayama, F. Bai, andN. Hara (1999). Granulocyte, granulocyte-macrophage, andmacrophage colony-stimulating factors can stimulate the in-vasive capacity of human lung cancer cells. Br. J. Cancer 79:40–46.

86. L. D. Yee and L. Liu (2000). The constitutive production ofcolony stimulating factor 1 by invasive human breast cancercells. Anticancer Res. 20:4379–4383.

87. H. Bando and M. Toi (2000). Tumor angiogenesis,macrophages, and cytokines. Adv. Exp. Med. Biol. 476:267–284.

88. B. Dankbar, T. Padro, R. Leo, B. Feldmann, M. Kropff, R. M.Mesters, H. Serve, W. E. Berdel, and J. Kienast (2000). Vas-cular endothelial growth factor and interleukin-6 in paracrinetumor–stromal cell interactions in multiple myeloma. Blood95:2630–2636.

89. C. Frelin, A. Ladoux, and G. D’Angelo (2000). Vascular en-dothelial growth factors and angiogenesis. Ann. Endocrinol.(Paris) 61:70–74.

90. H. Saji, M. Koike, T. Yamori, S. Saji, M. Seiki, K. Matsushima,and M. Toi (2001). Significant correlation of monocytechemoattractant protein-1 expression with neovascularization

P1: GDX



and progression of breast carcinoma. Cancer 92:1085–1091.

91. D. Toomey, C. Condron, Q. D. Wu, E. Kay, J. Harmey, P.Broe, C. Kelly, and D. Bouchier-Hayes (2001). TGF-beta1is elevated in breast cancer tissue and regulates nitric ox-ide production from a number of cellular sources duringhypoxia re-oxygenation injury. Br. J. Biomed. Sci. 58:177–183.

92. L. L. Thomsen and D. W. Miles (1998). Role of nitric oxidein tumour progression: Lessons from human tumours. CancerMetastasis Rev. 17:107–118.

93. L. C. Jadeski and P. K. Lala (1999). Nitric oxide synthase inhi-bition by N(G)-nitro-L-arginine methyl ester inhibits tumor-induced angiogenesis in mammary tumors. Am. J. Pathol.155:1381–1390.

94. R. Rajan, R. Vanderslice, S. Kapur, J. Lynch, R. Thompson,and D. Djakiew (1996). Epidermal growth factor (EGF) pro-motes chemomigration of a human prostate tumor cell line,and EGF immunoreactive proteins are present at sites ofmetastasis in the stroma of lymph nodes and medullary bone.Prostate 28:1–9.

95. G. D. Roodman (2001). Biology of osteoclast activation incancer. J. Clin. Oncol. 19:3562–3571.

96. H. Kodama, M. Nose, S. Niida, and A. Yamasaki (1991). Es-sential role of macrophage colony-stimulating factor in theosteoclast differentiation supported by stromal cells. J. Exp.Med. 173:1291–1294.

97. A. Grey, Y. Chen, I. Paliwal, K. Carlberg, and K. Insogna(2000). Evidence for a functional association between phos-phatidylinositol 3- kinase and c-src in the spreading responseof osteoclasts to colony-stimulating factor-1. Endocrinology141:2129–2138.

98. N. C. Hunt, Y. Fujikawa, A. Sabokbar, I. Itonaga, A. Harris,and N. A. Athanasou (2001). Cellular mechanisms of boneresorption in breast carcinoma. Br. J. Cancer 85:78–84.

99. J. M. Quinn, J. O. McGee, and N. A. Athanasou (1998). Humantumour-associated macrophages differentiate into osteoclas-tic bone-resorbing cells. J. Pathol. 184:31–36.

100. A. T. Mancino, V. S. Klimberg, M. Yamamoto, S. C. Manolagas,and E. Abe (2001). Breast cancer increases osteoclastogene-sis by secreting M-CSF and upregulating RANKL in stromalcells. J. Surg. Res. 100:18–24.

101. J. W. Pollard and L. Henninghausen (1994). Colony stim-ulating factor-1 is required for mammary gland develop-ment during pregnancy. Proc. Natl. Acad. Sci. U.S.A. 91:9312–9316.

102. P. E. Cohen, L. Zhu, and J. W. Pollard (1997). The absence ofCSF-1 in osteopetrotic (csfmop/csfmop) mice disrupts estrouscycles and ovulation. Biol. Reprod. 56:110–118.

103. A. V. Nguyen and J. W. Pollard (2002). Colony stimulatingfactor-1 is required to recruit macrophages into the mam-mary gland to facilitate mammary ductal outgrowth. Dev. Biol.247:11–25.

104. R. C. Humphreys, M. Krajewska, S. Krnacik, R. Jaeger, H.Weiher, S. Krajewski, J. C. Reed, and J. M. Rosen (1996).Apoptosis in the terminal endbud of the murine mammarygland: A mechanism of ductal morphogenesis. Development122:4013–4022.

105. L. M. Coussens, C. L. Tinkle, D. Hanahan, and Z. Werb (2000).MMP-9 supplied by bone marrow-derived cells contributes toskin carcinogenesis. Cell 103:481–490.

106. L. M. Coussens and Z. Werb (2001). Inflammatory cells andcancer: Think different! J. Exp. Med. 193:F23–26.

107. H. Kuper, H. O. Adami, and D. Trichopoulos (2000). Infectionsas a major preventable cause of human cancer. J. Intern. Med.248:171–183.

108. L. A. Garcia Rodriguez and Huerta-Alvarez, C. (2001). Re-duced risk of colorectal cancer among long-term users of as-pirin and nonaspirin nonsteroidal anti-inflammatory drugs.Epidemiology 12:88–93.

The Macrophage Growth Factor CSF1 in Mammary Gland Development and Tumor Progression

Documents

Transcript of The Macrophage Growth Factor CSF1 in Mammary Gland Development and Tumor Progression