ORIGINAL ARTICLE

Roger S. Jackson II . Omar E. Franco .

Neil A. Bhowmick

Gene Targeting to the Stroma of the Prostate and Bone

Received December 7, 2007; accepted in revised form February 18, 2008

Abstract Stromal–epithelial interactions mediated byparacrine signaling mechanisms dictate prostate devel-opment and progression of prostate cancer. The regu-latory role of androgens in both the prostate stromaland epithelial compartments set the prostate apart frommany other organs and tissues with regard to gene tar-geting. The identification of androgen-dependent pros-tate epithelial promoters has allowed successful genetargeting to the prostate epithelial compartment. Cur-rently, there are no transgenic mouse models availableto specifically alter gene expression within the prostatestromal compartment. As a primary metastatic site forprostate cancer is bone, the functional dissection of thebone stromal compartment is important for under-standing stromal–epithelial interactions associated withmetastatic tumor growth. Use of currently availablemethodologies for the expression or deletion of geneexpression in recent research studies has advanced ourunderstanding of the stroma. However, the complexityof stromal heterogeneity within the prostate remains achallenge to obtaining compartment or cell-lineage-specific in vivo models necessary for furthering ourunderstanding of prostatic developmental, benign,tumorigenic, and metastatic growth.

Key words prostate � stroma or stromal �mesenchymal � bone � osteoblasts � tissuerecombination xenografting � transgenic mice �conditional knock-out

Introduction

The role of mesenchymal tissues in the control of or-ganogenesis has been well established in many organsincluding the prostate (Wolff, 1968; Cunha, 1972; Slav-kin, 1974; Cunha et al., 1980; Kollar and Fisher, 1980;Cunha and Chung, 1981; Chung and Cunha, 1983;Chung et al., 1984; Saxen et al., 1986). The prostatedevelops from an ambisexual embryonic rudiment, theendodermal urogenital sinus (UGS). The prostatic ure-thra and bulbourethral glands in males, the lower va-gina and urethra in females, and bladder in both sexesalso develops from the UGS. Embryonic connectivetissue surrounding the UGS, referred to as urogenitalmesenchyme (UGM), expresses androgen receptors(ARs) and responds to fetal testicular androgens bypromoting epithelial development and differentiation.Embryonic development of the prostatic glandular ep-ithelium proceeds from the outward expansion of cordsof urogenital epithelium (UGE) from the UGS into theUGM resulting in the formation of prostatic buds. Theprostatic buds elongate and undergo branching mor-phogenesis and canalization resulting in the formationof prostatic ductal trees. In rodents, the prostatic ductaltrees organize into distinct lobes that include the ante-rior, dorsal–lateral, and ventral prostate. In contrast,the prostatic ductal trees in humans organize into asolid compact gland where distinct sets of ducts result inthe organization of the central, transitional, and pe-ripheral zones (reviewed in Thomson and Marker, 2006;Cunha, 2008, this issue).

Stromal tissues form much of the connective tissueand basement membrane support structures upon andwithin which prostate epithelia are dependent for sur-vival, growth, and differentiation. UGM-derived pros-tatic stroma specifies prostate epithelial identity andsubsequent differentiation into mature secretory epi-thelium (reviewed in this issue in Cunha, 2008;Matusik, et al. 2008). In turn, the developing prostaticepithelium induces the UGM-derived prostatic stroma

Roger S. Jackson II � Omar E. Franco �Neil A. Bhowmick ( .*)Department of Urologic Surgery, Vanderbilt-Ingram CancerCenter, Vanderbilt University Medical Center, Nashville TN37232-2765, USA.Tel: +1 615 343 7140Fax: +1 615 322 5869E-mail: neil.bhowmick@vanderbilt.edu

Differentiation (2008) DOI: 10.1111/j.1432-0436.2008.00273.xr 2008, Copyright the AuthorsJournal compilation r 2008, International Society of Differentiation

to undergo smooth muscle differentiation (Hayward etal., 1996b). Components of the adult prostatic stromainclude the basement membrane and extracellular ma-trix (ECM) that is produced by fibroblasts and smoothmuscle cells associated with the ductal tissue. Thestroma of the prostate also contains blood vessels,nerves, and infiltrating or recruited bone marrow-de-rived cells. Mesenchymally derived cells of the bone,namely osteoblasts and osteoclasts, are also of interestas bone is a frequent metastatic site for prostate cancer.The prostatic and bone stromal tissue components,however, are a mixture of yet undefined states of differ-entiation. It is undefined because few stromal cell- andtissue-specific markers are known. The knowledge ofthese specific markers would enable specific targeting ofgene expression or knock-down to a specific cell lineageor tissue compartment. Stromal–epithelial interactionsare mediated principally by epithelial and stromalsecreted cytokines, chemokines, and growth factors(Donjacour and Cunha, 1991). The biology of theECM, matrix degrading enzymes, as well as endothelialand immune cells contributes significantly to our un-derstanding of stromal–epithelial interactions and thetumor microenvironment. While these topics are notwithin the scope of this article, these are discussed inother reviews in this issue.

A particular focus of recent studies in prostate car-cinogenesis has been the functional dissection of thestromal compartment. Genetic targeting of the stromahas been made difficult by stromal heterogeneity. Thestroma for any given anatomical site of interest iscomposed of multiple types of stromal cells, while pre-dominated by either fibroblasts or smooth muscle cells,resulting in internal cellular heterogeneity. The typesand ratios of stromal cells present within an anatomicalsite of interest is regulated by the developmental stage,the state of differentiation of the surrounding cells andtissue, and the normal ongoing homeostatic responsesto local paracrine signaling, inflammation, and tissueremodeling and repair. While morphologically similar,stromal cells from a specific cell lineage (fibroblasts forexample) isolated from different organ or tissue siteshave distinct patterns of gene expression imposed bydevelopmental cell fate programming. Observed varia-tions in gene expression between colonies derived froma single stromal cell lineage, isolated from a specifictissue site, and grown in culture have demonstrated thepresence of multiple cell sub-populations with distinctphenotypic and functional characteristics. The detectionof multiple cell sub-populations within a given celllineage has been demonstrated ex vivo by various tech-niques including flow cytometry, immunohistochemis-try, immunofluorescence, and in situ hybridization.For example, fibroblasts localized at different levels orlocations within the dermis have been shown to havedistinct patterns of gene expression by microarray anal-ysis, and specific dermal fibroblast sub-populations are

differentially activated during the wound healing pro-cess (Schor and Schor, 1987; Irwin et al., 1994; Jelaskaet al., 1999; Chang et al., 2002; Sorrell and Caplan,2004; Nolte et al., 2007). Here we review the role ofstromal–epithelial interactions in prostate developmentand tumorigenesis as well as the methodology currentlyavailable to target gene expression for studies of theprostatic and bone stromal compartments.

Stromal–epithelial interactions in prostatedevelopment

Prostate development and function is tightly regulatedby various autocrine and paracrine factors that includecytokines and chemokines, growth factors like trans-forming growth factor-b (TGF-b), and endocrine hor-mones such as androgens (Hayward and Cunha, 2000;Montgomery et al., 2001). The action of androgens ismediated by the AR, a member of the steroid hormonereceptor superfamily. Testosterone upon cellular entryis converted into 5a-dihydrotestosterone (DHT) bythe enzyme, 5a-reductase. Upon DHT binding, ARbecomes phosphorylated and undergoes conformatio-nal changes that release it from heat shock proteins inthe cytoplasm. This allows AR to migrate into thenucleus where it interacts with androgen responsiveelements found in the promoters of androgen-regulatedgenes (Blok et al., 1996; Brinkmann et al., 1999; Peter-ziel et al., 1999; Maeda, 2001; Georget et al., 2002). ARin the prostatic epithelial cells (Cooke et al., 1991)up-regulates the expression of various proteins charac-teristic of the differentiated state including prostate-specific antigen (PSA) and probasin, expressed inhumans and rodents, respectively (Donjacour andCunha, 1993; Snoek et al., 1998; Zhang et al., 2004),(Table 1).

Androgens regulate prostate development in partthrough stromal–epithelial interactions. The androgenicand neonatal development of prostatic tissue is medi-ated by androgenic effects within the mesenchyme(Cunha and Chung, 1981; Cunha and Donjacour,1989). For example, androgens can induce stromalfibroblasts to express paracrine growth factors, likeFGF8 and IGF-1, that stimulate epithelial proliferation(Ruan et al., 1999; Gnanapragasam et al., 2002; Wuet al., 2007a). Androgens can also modulate the activityof other stromally produced paracrine factors, such asEGF, FGF7/KGF, FGF10, and TGF-a, upon theprostatic epithelium (Morton et al., 1990; Reiter et al.,1992; Yan et al., 1992; Bacher et al., 1993; Gray et al.,1995; Sokoloff et al., 1996; Thomson et al., 1997; Itohet al., 1998; Thomson and Cunha, 1999).

Tissue recombination experiments provide evidencethat paracrine signaling from the mesenchymal cellsto the epithelial cells in part controls prostate gland

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development. When allografted with wild-type UGM,prostatic glands with normal glandular architecture butlacking prostatic secretory function developed fromepithelia (expressing a nonfunctional AR) derived fromtesticular feminized (Tfm) mice (Sugimura et al., 1986;Cunha, 2008, this issue). The resulting prostatic glandswere equally sensitive to androgen ablation carried outby castration of the host mice, as compared with thecontrol tissue recombinations generated with wild-typemesenchyme and wild-type epithelia (Kurita et al.,2001). Reciprocal tissue recombinations using TfmUGM and wild-type epithelia failed to undergo pros-tatic development (Cunha, 2008, this issue). Together,these data established that stromal androgen respon-siveness is required for glandular development of theprostatic epithelium (Cunha and Lung, 1978; Donjac-our and Cunha, 1993). This suggests that the prostaticstroma plays an instructive role in glandular develop-ment and influences prostatic responsiveness to andro-gen ablation (Kurita et al., 2001; Cunha, 2008, thisissue).

Interestingly, castration induced regression of theprostate results in a detectable long-term increase inTGF-b expression (Kyprianou and Isaacs, 1989; Mart-ikainen et al., 1990; Shidaifat et al., 2007). Inductionof a wave of apoptotic death of the prostatic bloodvasculature occurs after castration and before theregression of the prostatic stromal and epithelial com-partments (Kyprianou and Isaacs, 1988; Isaacs et al.,1992, 1994; Furuya et al., 1995; Denmeade et al., 1996;Shabisgh et al., 1999; Shabsigh et al., 1999a, 1999b).Together, these data suggest a mechanism wherebyincreased TGF-b expression may contribute to theinitiation and maintenance of prostate regression. Fol-lowing androgen ablation, interaction of the TGF-band androgen signaling pathways most likely involvesautocrine and paracrine cross-talk involving multiple

cytokines and growth factors. Both cooperative andantagonistic interactions of androgen and TGF-b sig-naling pathways have been reported in the prostaticepithelia (Hayes et al., 2001; Kang et al., 2001; Chipuket al., 2002), suggesting a possible role for TGF-bsignaling in the regulation of androgen responsiveness.

TGF-b isoforms (TGF-b1, b2, b3) have long beenestablished as physiological regulators of prostategrowth because of their ability to inhibit cell prolifer-ation and mediate apoptosis (Kyprianou and Isaacs,1989; Martikainen et al., 1990). TGF-b isoforms exerttheir effects through binding to the TGF-b type IIreceptor (Tgfbr2) and subsequent recruitment of thetype I receptor (Tgfbr1) for down-stream cytoplasmicsignaling through multiple parallel signaling pathwaysincluding activation of p38, PI3K/AKT, RhoA/Rock,and SMADs (Massague and Gomis, 2006). SMAD sig-naling has also been associated with the transcriptionaldown-regulation of the mitogenic proto-oncogene,c-Myc (Alexandrow and Moses, 1995; Feng et al.,2002; Yagi et al., 2002). The TGF-� type III receptor(Tgfbr3), required for TGF-b2 signaling throughTgfbr2, is expressed by the prostatic stroma, but to alesser extent in the prostatic epithelia (Bhowmick et al.,2004a). The intracellular, extracellular, and microenvi-ronmental contexts determine the outcome of TGF-bsignaling in terms of the regulation of differentiation,growth (proliferation and apoptosis), and other bio-logical endpoints (Bierie and Moses, 2006; Stover et al.,2007).

The glandular compartment of the adult humanprostate (Fig. 1) consists of mature differentiated basaland secretory luminal epithelial cells with few neuroen-docrine cells. The epithelial cells are in close contactwith an underlying stromal compartment consistingmainly of differentiated smooth muscle cells in ECM.(For a review about prostate epithelial cell fates, see

Table 1 Prostate epithelial and stromal differentiation markers

Differentiation Marker Symbol(s) Compartment Reference

Androgen Receptor AR Epithelia Cooke et al. (1991)Cytokeratin 5 CK5 Basal epithelia Wang et al. (2001)Cytokeratin 8 CK8 Luminal epithelia Wang et al. (2001)Cytokeratin 14 CK14 Basal epithelia Wang et al. (2001)Cytokeratin 18 CK18 Luminal epithelia Wang et al. (2001)Desmin DES Myofibroblasts, smooth muscle Hayward et al. (1996b)Fibroblast Specific Protein-1 FSP-1, S100A4 Fibroblasts, myofibroblasts, and

smooth muscleBhowmick et al. (2004a)

p63 p63 Basal epithelia Wang et al. (2001)Prostatic-Acid Phosphatase PAP Differentiated prostatic epithelium Hayward et al. (1998)Probasin Pb Epithelia Zhang et al. (2004)Prostate-Specific Antigen PSA Epithelia Snoek et al. (1998)SM22 SM22 Myofibroblasts, smooth muscle Duband et al. (1993)Smooth Muscle Actin (alpha) aSMA Myofibroblasts, smooth muscle Tuxhorn et al. (2002a)Smooth Muscle MyosinHeavy Chain

SMMHC Myofibroblasts, smooth muscle Regan et al. (2000)

Tenascin TEN Myofibroblasts, smooth muscle Tuxhorn et al. (2002a)Vimentin Vim Fibroblasts, myofibroblasts Tuxhorn et al. (2002a)

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Matusik et al., 2008 and Abate-Shen et al., 2008, bothin this issue.) During prostate development, me-senchymal cells progressively differentiate into smoothmuscle cells that constitute the adult prostatic stroma.The smooth muscle cells are thought either to differen-tiate directly from fibroblasts or in a step-wise progres-sion from fibroblasts to myofibroblastic cells to well-differentiated smooth muscle cells (Hayward et al.,1996b, 2006). The maturation of the stroma supportsdifferentiation of proliferative immature epithelia intowell-differentiated, quiescent adult glandular epithelia.As both prostate epithelial and stromal cells express AR(Prins et al., 1991), the maintenance of the growth-qui-escent state of the adult prostate is directly regulated inpart by androgen signaling and reciprocal stromal–epithelial interactions.

Markers frequently used to assess prostate epithelialdifferentiation (Table 1) include p63 and prostatic acidphosphatase, as well as cytokeratins 5, 8, 14, and 18(Wang et al., 2001, 2005a). Assessment of prostaticstromal differentiation is more complex as knownstromal differentiation markers (Tables 1 and 2) are

often expressed in an increasing or decreasing manneracross a continuum of lineage differentiation. For ex-ample, prostatic fibroblasts express fibroblast specificprotein-1 (FSP-1, S100A4) and vimentin (Sugimoto etal., 2006). Myofibroblasts also express FSP-1 and vi-mentin as well as some smooth muscle-specific proteins,such as desmin, a-smooth muscle actin (aSMA), andtenascin (Hayward et al., 1996b; Tuxhorn et al., 2002a).Mature differentiated prostatic smooth muscle cells ex-press these and other smooth muscle-specific proteins,such as SM22 and smooth muscle myosin heavy chain(SMMHC), and little to no fibroblast-specific genes likeFSP-1 or vimentin (Duband et al., 1993).

Stromal–epithelial interactions in primaryprostate cancer

Accumulation of genetic alterations in adult prostaticepithelium promotes carcinogenesis through aberrant

Fig. 1 Prostate epithelial interactions with prostate stromal com-partments. Glandular epithelium (basal, luminal, and occasionalneuroendocrine cells) of the normal human adult prostate is in closecontact with the underlying stromal compartment consisting pre-dominately of ECM (collagen, fibronectin, etc.), differentiatedsmooth muscle cells, as well as occasional fibroblasts, myofibro-blasts, and blood vessels. In prostate cancer, de-differentiation ofthe prostatic smooth muscle cells into fibroblasts promotes de-differentiation, proliferation, and stromal invasion of the prostaticepithelium. This results in a vicious cycle of disease progression,which includes localized increases in angiogenesis to/from the tu-mor as well increased recruitment of myeloid-lineage hematopoieticcells. Differentiation markers for which there are available Cre micetargeting prostate stromal compartments are listed. ECM, extra-cellular matrix.

Table 2 Cre mice available for targeting mesenchymal/stromalcompartments of the prostate and bone

Mouse line Targeted tissue forCre expression

References

CD11b-Cre Macrophages, monocytes,osteoclasts, granulocytes

Ferron and Vacher(2005)

Col1a1-Cre Fibroblasts,myofibroblasts, smoothmuscle, osteoblasts,chondrocytes

Dacquin et al. (2002),Liu et al. (2004),Kim et al. (2004)

Col1a2-Cre/Cre-ERT

Fibroblasts,myofibroblasts, smoothmuscle, osteoblasts,chondrocytes

Zheng et al. (2002),Florin et al. (2004)

Col2a1-Cre/Cre-ERT

Chondrocytes Long et al. (2001),Sakamoto et al. (2005),Nakamura et al. (2006)

CTSK-Cre Osteoclasts Chiu et al. (2004)Dermo1-Cre Chondrocytes, osteoblasts Yu et al. (2003)FSP1-Cre Fibroblasts,

myofibroblasts, smoothmuscle, osteoblasts,chondrocytes,macrophages

Iwano et al. (2001,2002),Bhowmick et al.(2004a);Inoue et al. (2005)

OC-Cre Chondrocytes, osteoblasts Zhang et al. (2002)Prx1-Cre Chondrocytes Logan et al. (2002)SMA-Cre Myofibroblasts, smooth

muscleMiwa et al. (2000),Wu et al. (2007b)

SM22-Cre/Cre-ERT

Smooth muscle Kuhbandner et al.(2000),Holtwick et al. (2002)

SMMHC-Cre Smooth muscle Regan et al. (2007)Sox9-Cre Chondrocytes, osteoblasts Akiyama et al. (2005)Tie1-Cre Endothelial cells Song et al. (2007)Tie2-Cre Endothelial cells,

MacrophagesKisanuki et al. (2001)

TRAP-Cre Chondrocytes, osteoclasts Chiu et al. (2004)

FSP, fibroblast specific protein; SMA, smooth muscle actin;SMMHC, smooth muscle myosin heavy chain.

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changes in gene expression supporting increased epithe-lial proliferation and evasion from apoptosis. For in-stance, over-expression of c-Myc (Fleming et al., 1986;Matusik et al., 1987; Jenkins et al., 1997; Ellwood-Yenet al., 2003) and loss of PTEN (Cairns et al., 1997) arethought to be early events in prostate carcinogenesis incomparison to the later stage loss of p53 and pRb(Jenkins et al., 1997; Jarrard et al., 2002; Dong, 2006).Normal prostate epithelial and stromal cells expressTGF-b type I and II receptors (Royuela et al., 1998).However, the loss of expression of the TGF-b typeI and II receptors in the prostatic epithelium correlateswith increased tumor grade in human prostate cancertissues and poor prognosis (Kim et al., 1996, 1998; Tuet al., 2003). Loss of TGF-b type II receptor in thestroma correlates with poor patient prognosis and thepresence of tumor-associated macrophages in coloncancer patients (Bacman et al., 2007), suggesting thatstromal loss of Tgfbr2 expression may contribute to theprogression of other cancers including those of theprostate. Prostatic intraepithelial neoplasia (PIN), aproposed precursor for prostatic adenocarcinoma, isalso reported to have similar alterations (Bostwicket al., 1995, 1996; Sakr and Partin, 2001).

Just as the stroma has an important role in regulatingthe development and maturation of the prostatic epi-thelium, the stroma too plays a key role in the suppres-sion of epithelial transformation. Normal stromal cellshave been reported to convert malignant epithelia tomorphologically benign lesions in vitro (Cooper andPinkus, 1977; Hayashi and Cunha, 1991). Changes ingene expression in normal stromal cells, however, canpromote malignancy in the neighboring unaltered epith-elia (Nakamura et al., 1997; Barcellos-Hoff and Ravani,2000) and malignant progression in neighboring initi-ated epithelia (Olumi et al., 1999; Hayward et al., 2001;Phillips et al., 2001). For example, we have previouslydemonstrated that the knock-out of Tgfbr2 expressionin the stroma promotes the formation of PIN lesionsand supports the transformation of adjacent epitheliumin prostate and forestomach tissues (Bhowmick et al.,2004a). Another example comes from recent work pub-lished by Hill et al., 2005a, 2005b in which the inacti-vation of pRB in the prostatic epithelium of geneticallyengineered mice resulted in an initial induction of p53in the epithelium and underlying stroma followed by aselective loss in stromal p53 expression. This loss instromal p53 expression resulted in increased proliferationof the prostatic stroma and subsequent increased tumoraggressiveness. In this study, lack of histologic observa-tion of basement membrane breakdown or of stromalcells co-expressing both epithelial and fibroblastic mark-ers supported a paracrine stromal response mechanismas opposed to an epithelial to mesenchymal transition(EMT) (Hill et al., 2005b). A similar promotion in cancerprogression and aggressiveness was observed by Kiariset al., 2005 in tissue recombination studies in which

MCF7 mammary epithelial cells were recombined andgrafted with p531/1 or p53� /� stroma.

The evolution of prostate cancer results ultimately inthe establishment of a vicious cycle of disease progres-sion and subsequent metastasis (Sung and Chung, 2002;Cunha et al., 2003; Bhowmick et al., 2004b; Bhowmickand Moses, 2005; Bierie and Moses, 2005). Loss ofdifferentiation in genetically initiated prostatic epitheli-um results in altered paracrine signaling from theepithelium to the underlying differentiated adult pros-tatic stroma. In turn, the prostatic stromal cells undergoa reversal in differentiation to become reactive myo-fibroblasts, promoting malignant progression throughaltered stromal to epithelial paracrine signaling (Wonget al., 2003). Furthermore, neoplastic prostatic epithe-lial cells may be able to acquire the ability to assume afibroblastic phenotype by undergoing EMT, similar tothat demonstrated in breast cancer biopsy tissues(Petersen et al., 2003). Another alternative mechanismmight be that loss of epithelial and stromal differenti-ation in the prostate leads to the recruitment of bonemarrow-derived cells that subsequently take up resi-dence and differentiate into myofibroblasts in thestroma (Lincoln et al., 1997; Brittan et al., 2002, 2005;Direkze et al., 2003; Hashimoto et al., 2004; Hayashidaet al., 2005; Jabs et al., 2005; Mori et al., 2005; Yam-aguchi et al., 2005; Haudek et al., 2006; Asawa et al.,2007; Bellini and Mattoli, 2007; Dupuis et al., 2007; Liet al., 2007; Morimoto et al., 2007).

Stromal myofibroblasts have been referred to bymany names including ‘‘tumor-associated stroma’’(Mueller and Fusenig, 2004; Chung et al., 2005), ‘‘re-active stroma’’ (Tuxhorn et al., 2001, 2002a, 2002b),and ‘‘carcinoma-associated fibroblasts’’ (CAFs) (Olumiet al., 1999; Cunha et al., 2002, 2003). CAFs have beendemonstrated to secrete altered levels of a number ofcytokines and growth factors including TGF-b (SanFrancisco et al., 2004; Chung et al., 2005; Joesting et al.,2005; Orimo et al., 2005; Kalluri and Zeisberg, 2006).CAFs derived from human prostate tumors are able toelicit malignant progression in the immortalized non-tumorigenic human prostatic epithelial cell line, BPH-1(Olumi et al., 1999). This tumorigenic effect can be ab-rogated by treating animals with a TGF-b neutralizingantibody (2G7) (Ao et al., 2007). Together, these dataprovide compelling evidence that stromal–epithelialinteractions within the prostate microenvironment regu-late and modulate the behavior of normal and malig-nant prostate epithelial cells.

Bone stromal interactions with metastaticprostate cancer

During development, embryonic bone mesenchymalcells differentiate into hematopoietic stem cells (whichgive rise to the lymphoid and myeloid lineage cells of

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the blood) or bone-lineage stromal cells (Marieb, 1998).Derived from differentiated macrophages (a myeloidlineage of hematopoietic cells), osteoclasts are foundlining the inner surfaces within trabecular bone andfunction to actively resorb bone (Roodman, 2006).Bone-marrow fibroblasts line the inner surface of theblood marrow cavity. Chondrocytes, which producecartilage, are found in cartilaginous tissues includingthe growth plates of long bones in adolescents (Marieb,1998). Osteoblasts, which actively form bone, are foundlining the inner surfaces of trabecular bone (Marieb,1998). Chondrocytes and osteoblasts are thought todifferentiate from a common bone mesenchymal/stro-mal-lineage precursor (Romero-Prado et al., 2006).

Bone is the most frequent site of prostate cancer me-tastasis and the major site of tumor burden in mostpatients with advanced disease. Thereby, complicationsarising from metastasis of prostate cancer to the bone ismost often the primary reason for the morbidity andmortality of prostate cancer patients (Chirgwin andGuise, 2007; Kingsley et al., 2007a, 2007b). Expressionof bone specific stromal proteins in organ confinedprostate cancer, a process termed osteomimicry, maycontribute to the ability of migratory prostate tumorcells to successfully seed distant bone metastatic sites(Koeneman et al., 1999; Zayzafoon et al., 2004). Fur-ther, the ability of the bone stroma to respond toandrogens may contribute to the establishment of met-astatic prostate tumors in the bone. AR expression isdetectable in multiple cell types within the bone micro-environment including bone marrow stromal fibro-

blasts, chondrocytes, endothelial cells, osteoblasts,osteoclasts, and osteocytes. It is not surprising, there-fore, that the expression of multiple genes expressed inthe bone is regulated by androgen signaling and thatandrogens play an important role in the regulation ofbone remodeling (Wiren, 2005; Xu et al., 2007).

When tumor cells metastasize to bone, another vi-cious cycle is created between the metastatic tumor cellsand normal host bone cells (Mundy, 2002), specificallywith osteoclasts and osteoblasts (Fig. 2). Tumor cellsproduce paracrine factors that can stimulate osteoclastor osteoblast activity. Activated osteoclasts in turncause tumor cells to alter gene expression, behave moreaggressively, and cause destructive local bone osteolysis(Guise and Mundy, 1998; Mundy and Yoneda, 1998;Yin et al., 1999; Mundy, 2002). TGF-b, the most abun-dant growth factor present in bone, is stored in latentform in bone matrix and released in active form duringosteolytic bone resorption (Pfeilschifter and Mundy,1987). Release of TGF-b by osteolysis enhances expres-sion of parathyroid hormone related protein (PTHrP)by tumor cells, which in turn enhances osteoclast acti-vation and subsequent osteolysis as observed in studiesof metastatic breast cancers (Guise et al., 1996; Yinet al., 1999). Similar to breast cancer cell lines, somehuman prostate cancer cell lines can establish osteolyticbone lesions, such as PC3 cells (Sanchez-Sweatmanet al., 1998). In general, prostate cancer at the time ofthe initial establishment of the tumor cells in the boneresults in transient osteolysis followed by predominantosteoblast activation. Activated osteoblasts in turn

Fig. 2 Prostate tumor cell interactions with bone stromal compart-ments. The activities of bone resorption by osteoclasts and boneformation by osteoblasts are tightly coordinated. In bone, osteo-clasts and osteoblasts are frequently localized in close proximity toeach other and interact via paracrine signaling. Infiltration of met-astatic prostate tumor cells begins a vicious cycle, mediated bycross-talk of paracrine signaling intermediates, such as TGF-�, Wnt

and DKK1, Sonic Hedgehog (SHH), Endothelin-1 (ET-1), andRank/RankL. Pro-osteolytic cancers stimulate osteoclast differen-tiation and activity resulting in bone destruction. Pro-osteoblasticcancers stimulate osteoblast differentiation and activity resulting inbone formation of lesions of weaker woven bone within thetrabecular bone. Differentiation markers for which there are avail-able Cre mice targeting bone stromal compartments are listed.

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cause the tumor cells to alter gene expression, behavemore aggressively, and cause destructive formation ofweaker woven bone. Examples of human prostate can-cer cell lines that establish osteoblastic lesions includeLNCaP-C4-2B (Lin et al., 2001) and LuCaP 23.1 (Liuet al., 1996). CWR22 cells have been reported to estab-lish both osteolytic and osteoblastic lesions (Andersenet al., 2003). The osteolytic to osteoblastic transition ofprostate cancer bone metastasis is thought to be regu-lated by tumor and bone cell produced factors includingTGF-b, Wnt family ligands, and Wnt inhibitors (Kellerand Brown, 2004; Hall et al., 2005, 2006; Logothetisand Lin, 2005; Hall and Keller, 2006). Thereby, hijack-ing of paracrine signaling between osteoclasts andosteoblasts by the tumor cells may contribute to thesurvival and expansion of metastatic lesions in the bone.

Targeting gene deletion or expression to stromalcompartments of the prostate and bone

Stromal–epithelial interactions are essential for normalprostate development and contribute significantly to theprocess of prostate carcinogenesis at the primary andmetastatic sites. Research over the past four decades haselucidated much detail about the gene expression andsignal transduction of normal and neoplastic epithelia;however, much less is known about the stromal com-partment. The accumulation of data supporting theideas of cancers as ‘‘wounds that do not heal’’ (Dvorak,1986) and the ‘‘seed and soil’’ mechanism for cancermetastasis (Paget, 1889; McCawley and Matrisian,2001; Fidler et al., 2002) has also illuminated the needto increase our understanding of normal tissue andtumor microenvironments (Matrisian et al., 2001;Cunha and Matrisian, 2002).

Many mouse models are available for the study ofprostate cancer and prostate cancer metastasis (re-viewed in Singh and Figg, 2005). However the utility ofthese models to study stromal–epithelial interactionsand the tumor microenvironment is often severely lim-ited. Prostate epithelial and/or stromal cells (with orwithout genetic modification) can be injected orthotop-ically into the lobes of the mouse prostate. Left ven-tricular intra-cardiac and intra-tibial injections can beaccomplished and used to study bone metastasis (Wanget al., 2005b). The characteristics of a particular pros-tate cancer cell line, however, will ultimately determinethe utility of these models in terms of viable take ratesto the bone. Metastatic progression of prostate cancerin bone may also be studied using a model in whichbone tumors are established by grafting prostate cancerepithelia or stroma along with pieces of human fetalbone into male severe combined immunodeficient(SCID) mice. Establishment of prostate cancer cells inbone can be monitored in vivo through utilization of

imaging modalities such as X-ray, micro-computed to-mography (microCT), magnetic resonance imaging(MRI), and bioluminescence. Taken together, the low-take rates of the models preclude their more wide-spread use in experiments aimed at studying changes ingene expression in the prostatic epithelial and prostaticor bone stromal compartments (Nemeth et al., 1999).

Transgenic approaches for modulation of gene ex-pression in prostate epithelia are well established. Inaddition, multiple prostate cancer transgenic mousemodels have been generated and reviewed previously(Kasper and Smith, 2004; Abdulkadir and Kim, 2005;Kasper, 2005). Methods for in vivo targeted geneexpression or deletion in stromal compartments asopposed to epithelial compartments, however, havebeen developed more recently taking advantage ofrapidly developing improvements in conditional Cre/LoxP transgenic mice and RNA interference (RNAi).

Conditional gene knock-out in mice

Targeted gene insertion or deletion by homologous re-combination into the mouse genome (Fig. 3A) has beencrucial to the elucidation of the functional roles for en-dogenously expressed genes and the signaling networksto which they contribute. While specific for a gene locusof interest, constitutive knock-out of a gene(s) of inter-est frequently results in embryonic lethality where thegene(s) are critical for one or more stages of develop-ment. Introduction of the use of Cre and FLPe re-combinase mediated excision of loxP or FRT flankedexpression cassettes, respectively, increased the abilityto obtain the desired mouse model through improvedyield by reducing lethality phenotypes (Branda andDymecki, 2004; Sorrell and Kolb, 2005; Feil, 2007).Adenoviral Cre (Ad-Cre) and cell-permeable Cre areeffective measures for the in vitro knock-out or inver-sion of floxed genes or gene cassettes (Anton and Gra-ham, 1995; Jo et al., 2001; Joshi et al., 2002; Patsch andEdenhofer, 2007). Utility of Ad-Cre and cell-permeableCre in vivo is limited to accessible compartments wherenonspecific transduction of Cre into adjacent tissueswill not hamper the experimental outcome. Crossing ofmice bearing floxed allele(s) to mice expressing the Cretransgene improved knock-out cell-specificity, but wasdependent on the time and regulation of promoter ac-tivity (Fig. 3B). Examples include androgen-dependentpromoters active in the prostate epithelia such asNKX3.1-Cre (M. Shen, personal communication,UMDNJ-Robert Wood Johnson Medical School) andARR2Pb-Cre (based on the probasin promoter) (Wuet al., 2001), which are expressed early or late in pros-tate development, respectively. As no prostate stromal-specific promoters are yet available, various stromal

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promoters have been used with limited success in pros-tate studies.

Conditional knock-out utilizing stromal specificcre mice

Molecular regulation of Cre expression has allowed thecreation of more specific and informative mouse mod-

els. Developmental stage-specific gene promoters andtetracycline regulated (TET-on or TET-off) promotershave been used successfully to regulate the temporalinduction of expression of a transgene of interest in vivo(Fig. 3). Examples of such transgenes include Cre (Le-wandoski, 2001) and targeting inserts for RNA inter-ference (RNAi) mediated gene knock-down (Dickinset al., 2007). Similarly, use of tissue-specific promotersallows for restricted spatial expression to the compart-ment (tissue or organ) or cell lineage of interest, such asthe prostate or bone stroma (Table 2). Promoters forcollagen type 1 a 1 (Col1a1, 2.3 kb, or 3.7 kb promot-ers) (Dacquin et al., 2002; Kim et al., 2004; Liu et al.,2004), collagen type 1 a 2 (Col1a2) (Zheng et al., 2002;Florin et al., 2004), and FSP-1/S100A4 (Iwano et al.,2001, 2002; Bhowmick et al., 2004a; Inoue et al., 2005)can be used to target gene deletion or expression tofibroblasts, myofibroblasts, and smooth muscle cells(which constitute the stromal compartment) of theprostate. Fibroblasts can also be targeted utilizing thevimentin promoter and non-cardiac smooth muscle cellsusing the promoters for aSMA (Miwa et al., 2000; Wuet al., 2007b), SM22/transgelin (Kuhbandner et al.,2000; Holtwick et al., 2002), and SMMHC (Reganet al., 2000).

FSP-1 is expressed early in development at embry-onic day 9 (Iwano et al., 2001, 2002) and is inherentlyupstream of the mesenchymal differentiation lineagebeing expressed in precursor cells of the hematopoieticsystem, osteoblasts, and the mesenchyme of nearly ev-ery tissue examined including the prostate (Klingelhoferet al., 1997; Bhowmick et al., 2004a). The wide distri-bution of FSP-1 expression enables the study of the roleof stromal genes in many tissues. Of interest, the FSP-1gene is not expressed in the lineage of all cells of theprostate stroma (or other tissues), but rather a fractionclose to 30%–50% of the mature stromal cells. Thisobservation highlights the heterogeneity of the stromalcompartment within organs and enables the study ofjuxtacrine signaling within the stromal compartment.Importantly, FSP-1 promoter regulated gene targetingis an example of how targeting by other familiar stromalpromoters such as vimentin or SM22 will not be tissuespecific, but also only affect a fraction of the stromalcompartment (Sugimoto et al., 2006). The latter traitmay enable mouse viability from redundancy by otherstromal cells not affected. Expression of FSP-1 visual-ized by immunofluorescence of a green fluorescenceprotein (GFP) reporter in tissues including the bladder,prostate, and uterus illustrates both stromal specificityand stromal heterogeneity (Fig. 4) (Bhowmick et al.,2004a).

Previously, we generated a conditional mesenchymal(stromal) Tgfbr2 knock-out mouse using FSP-1 Cre-me-diated recombination (Tgfbr2fspKO) (Bhowmick et al.,2004a). While prostates from control Tgfbr2floxE2/floxE2

mice were morphologically normal, prostates from

Fig. 3 Conditional gene knockout in mice. (A) Integration of a genetargeting construct into a genomic locus by homologous recombi-nation and recombinase-mediated excision. A typical gene knock-out targeting construct has the following features: a region of DNAfrom a gene locus of interest containing one or more exons and thestart codon(s) flanked by loxP sites (referred to as being ‘‘floxed’’), anegative selection marker for integration such as the thymidinekinase (TK) cassette, and a positive selection marker for integrationsuch as the neomycin resistance gene (NEO) flanked by FRT sites(referred to as being ‘‘flrted’’). The linearized targeting construct isintroduced into mouse embryonic stem cells by electroporation ormicroinjection. The construct integrates into the genome by ho-mologous recombination resulting in loss of the negative selectionmarker. The positive selection marker is excised by transfection ofFLPe recombinase, which specifically recognizes FRT sites. Sub-sequently, the ES cells are placed in blastocysts and implanted intoa pseudo-pregnant female mouse resulting in the birth of micebearing the desired floxed allele. Introduction of Cre recombinase,which specifically recognizes loxP sites, will result in excision of thefloxed allele. (B) Generation of conditional knockout mice by se-lective breeding. Conditional knockout of gene expression in mice isaccomplished through the mating of a mouse bearing two copies ofthe floxed allele with a mouse expressing Cre recombinase driven bya gene promoter expressed in a developmental stage specific, tissuespecific, and/or temporally restricted manner. For example, themating of a FSP-1-Cre mouse with a floxed mouse will result in aproportion of the offspring having the desired FSP-1 directedknockout of the gene of interest (FSP-KO) in FSP-1 expressingstromal cells.

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Tgfbr2fspKO mice developed stromal and epithelial hy-perplasia with foci of hyperchromatic nuclei with atypiaby 5–7 weeks of age. These PIN lesions occurred pre-dominantly in the anterior and dorso-lateral lobes ofthe prostate. The Tgfbr2fspKO mice lacked nuclear phos-phorylated Smad2 immunostaining indicative of loss ofTGF-b signaling, as compared with floxed controls.Invasive squamous cell carcinoma of the forestomachconsistently developed in the Tgfbr2fspKO mice, whichdie prematurely by 8 weeks of age. The PIN lesions werereported to result from the reduction in the expressionof p21 and p27 (cdk inhibitors) and an increase in thelevels of c-Myc and phospho-c-Met receptor (deter-mined to be a consequence of increased HGF secretionby the stroma). In addition, the forestomach epithelialtissues of the Tgfbr2fspKO mice developed squamous cellcarcinoma through a presumed similar mechanism asthe pre-neoplastic lesions of the prostate. In summary,these studies demonstrated that blocking TGF-b sig-naling in the stromal compartment relieved the cell cycleinhibition in the epithelium and enabled proliferationand transformation of the adjacent epithelial compart-ment in prostate and forestomach tissues. Because ofthe early lethality of the Tgfbr2fspKO mouse model, ithas proven to be useful in studying initiating factorsinvolved in tumorigenesis and less useful for studyingsubsequent tumor progression. Tissue rescue of theTgfbr2fspKO prostates under the renal capsule ofsyngenic mice for 7 months have shown further pro-gression of the PIN lesions to adenocarcinoma (unpub-lished data).

If genes essential in early development are alteredusing a promoter driving Cre expression, such as FSP-1-Cre, early lethality may result in the mouse modelsoften precluding analysis of the tissue of interest. Suchearly developmental gene promoters, not surprisingly,have broader cell specificity. Unpublished observations

suggest additional FSP-1-Cre activity in the bone,which is of interest as osteoblasts are of mesenchymalorigin. The Col1a2-Cre mice, developed by de Crom-brugghe and colleagues, is expressed by many fibrob-lastic cell types (more restricted than FSP-1) and havethe added advantage of expressing a Cre-ER fusionprotein (Zheng et al., 2002). The modified estrogen re-ceptor provides a tight tamoxifen-regulation of Cre nu-clear localization, thus allowing both temporal andtissue-specific Cre activity. Because tamoxifen is onlyneeded briefly for Cre-mediated DNA recombinationactivity, any unwanted side-effects of tamoxifen can beavoided if the following experiments are performedabout 5–7 days following Cre activation. While use oftamoxifen to activate Cre-ER-mediated gene knock-outin unborn fetal mice is routinely successful, the abort-igenic effects of tamoxifen may hamper the ability toobtain live born mouse pups (Kuhbandner et al., 2000;Zheng et al., 2002; Nakamura et al., 2006). Adminis-tration of progesterone with tamoxifen may improveyield of living Cre-ER knock-out mice by helping tocounteract the abortigenic effects of tamoxifen (Nak-amura et al., 2006). However, the limiting factor inmore widespread use of Cre-ER is that there are only afew Cre-ER mouse models currently available for tar-geting the stromal compartment.

Mesenchymal derivatives of bone may also be tar-geted for genetic deletion or expression (summarized inTable 2). Chondrocytes and osteoblasts, which consti-tute bone stroma, can be targeted using Col1a1(Dacquin et al., 2002; Kim et al., 2004; Liu et al.,2004), Dermo-1 (Yu et al., 2003), FSP-1 (Iwano et al.,2001, 2002; Bhowmick et al., 2004a; Inoue et al., 2005),and Sox9 promoters (Akiyama et al., 2005). The pro-moter for collagen type 2 a 1 (Col2a1) can be used totarget chondrocytes as Col2a1 is not reported to be ex-pressed by osteoblasts or osteoclasts (Long et al., 2001;

Fig. 4 Fluorescent detection of FSP-1 expression in the stromalcompartments of multiple tissues of the bladder, prostate, anduterus. GFP expression driven by the FSP-1 promoter (green)is visible in these paraffin sections, nuclear counterstained with

Hoechst 33258 (blue). Importantly, note that a fraction of thestromal compartment has FSP-1 expression. The epithelial (E) andstromal (S) compartments are indicated.

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Sakamoto et al., 2005; Nakamura et al., 2006). Thepromoter for Prx1, a marker of the undifferentiatedosteoblast precursor, has been used for Cre-mediatedknock-out of gene expression in chondrocytes (Loganet al., 2002). The promoter for Osteocalcin (OC), amarker of differentiated osteoblasts positively regulatedby Runx2, has been reported to allow Cre-mediatedknock-out of gene expression in chondrocytes andosteoblasts (Zhang et al., 2002). Osteoclasts have beentargeted using promoters including Cathepsin K(CTSK) and Tartrate Resistant Acid Phosphatase(TRAP) (Chiu et al., 2004). The CD11b-Cre mice tar-gets cells of myeloid origin, which include osteoclasts,granulocytes (basophils, eosinophils, and neutrophils)and monocyte/macrophage lineages (Ferron and Vac-her, 2005). Endothelial cells can be targeted using Tie1(Song et al., 2007) or Tie2 promoters (Kisanuki et al.,2001), the latter of which also has been reported to beexpressed in macrophages. The genetic parallels of thebone and prostate stroma associated with gene targetingmay suggest similarities in biological regulation andthus is likely not coincidental that prostate cancermetastasizes to the bone.

Conditional knock-out utilizing RCASTVA/TVB mice

A potentially cost effective and efficient alternative toconventional mouse models, that frequently require ex-tensive breeding of transgenic and conditional gene al-terations in order to study synergistic interactions, is theutilization of the RCAS system for targeted gene ex-pression. Developed originally by Hughes and col-leagues and later adapted by the Varmus and Hollandlaboratories, the RCAS system takes advantage of ex-pression of TVA, a membrane-bound receptor for theavian leukosis virus sub-group A (ALV-A) retroviruses,not expressed in rodents or humans, encoded by theTVA gene (Bates et al., 1993; Federspiel et al., 1994;Holland et al., 1998a, 1998b; Holland and Varmus,1998; Fisher et al., 1999). Ectopic expression of theTVA gene into mammalian cells permits ALV-A infec-tion, viral entry and chromosomal integration, and ex-pression of genes carried by RCAS vectors. High-titerviral propagation can occur in the packaging avian cellline, DF-1 (Himly et al., 1998; Schaefer-Klein et al.,1998) or chicken embryonic fibroblasts (CEFs). AsALV-A viruses are not infectious to mammalian cells,this prevents cell-to-cell spread of the virus and reducedprobability of an immune response by the host becausethe targeted cells of interest are not producing viralpackaging proteins.

Because of the poor expression of ALV-A env gene ininfected TVA1 mammalian cells, the cells remain sus-ceptible to super-infection with RCAS vectors, allowing

for the transfer of multiple genetic lesions and/or histo-logic markers into a single TVA1 cell (Holland et al.,1998a, 1998b; Holland and Varmus, 1998). Important-ly, successful ALV infection has been possible only inreplicating cells until the recent development of lenti-viral counterparts that overcome the barrier of infectionto low replicative target cells (Naldini et al., 1996; Lewiset al., 2001). Alternatively, direct introduction of theavian packaging cell line, DF-1, into the desired site ofinfection can be utilized as the DF-1 cells produce freshvirus for a short period of time (3–4 days) and then dieoff. This method has been used successfully for infectingcells in the brain and bone marrow in the past (Hughesand Crittenden, 1991). Many RCAS viral vectors havebeen developed to express exogenous genes in ALV-Ainfected mammalian cells (Federspiel and Hughes,1997). These include the RCASBP(A) (RCAS, Bryan-RSV pol, sub-group A) vector that can accommodatean insert up to 2.5 kb and the RCASBP(B) (RCAS,Bryan-RSV polymerase, sub-group B) vector, whichcan accommodate a much larger insert. Virions pro-duced from RCASBP(A) and RCASBP(B) can recog-nize only TVA and TVB expressing cells, respectively.Because an exogenous promoter is utilized to drive ex-pression of genes of interest in the RCAS system, levelsof expression can be enhanced using promoters such asCMV. Alternatively, a promoter of higher tissue spec-ificity can be used to drive the gene of interest. For ahypothetical example, a TVA transgenic mouse drivenoff the FSP-1 promoter can be infected with an RCA-SBP(A) virus with a gene of interest driven by theCollagen 1a1 promoter. This was only hypothetical,because no stromally directed TVA/TVB have beenreported.

A number of laboratories have produced mouse linesexpressing TVA under a variety of tissue-specific pro-moters, making it possible to direct the expression of anRCAS vector to a specific cell or tissue type. The b-actinTVA mouse strain, which expresses TVA in all tissues,can enable preliminary experiments to test infectivity ofthe RCAS virus, expression of the gene of interest, andany promising phenotypes (Federspiel et al., 1996). Theb-actin TVA line is also a good source of primary cells/cell lines that can be efficiently infected in culture. No-tably, the b-actin TVA transgenic mice are in the C57/Bl6 background (of interest to those wanting to crossinto their mouse line of interest or those wanting toperform adoptive transfer experiments). Additionalpromoter driven TVA mouse lines and the tissues theytarget include: albumin (hepatocytes) (Lewis et al.,2005), a-actin (skeletal muscle) (Federspiel et al., 1994),BSP (bone) (Li et al., 2005), DCT (melanoblasts) (Dunnet al., 2000), elastase (pancreas) (Lewis et al., 2003),GFAP (astrocytes) (Holland and Varmus, 1998), GP-Iba (megakaryocyte lineage) (Murphy and Leavitt,1999), keratin 5 (keratin 5 positive cells such as ovar-ian surface epithelial cells) (Orsulic et al., 2002), MMTV

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(mammary gland) (Du et al., 2006), and nestin promot-ers (glial progenitors) (Holland et al., 1998a, 1998b).Utility of this technology can be further enhancedthrough the creation of additional TVA expressing micedriven by additional epithelial or stromal compartmentspecific gene promoters.

Tissue recombination grafting–transgenictissue rescue and compartment-specific genemodulation

Manipulation of gene expression or signaling activity intransgenic mice often impairs viability and lifespan.Therefore, cells and tissues from these mice needed forexperiments may not be available at the desired adultstage of development. A tissue of interest can be iso-lated from the host before death, for example the pros-tate from an adolescent male mouse, and rescued bysurgical implantation under the renal capsule of a 7–10weeks old normal adult male syngenic athymic nude(lacking T cells), or SCID mouse (lacking B cells andT cells) (Hayward et al., 1997; Day et al., 2002; Hay-ward, 2002). [An overview of the renal capsule graftingprocedure is available online (http://mammary.nih.gov/tools/mousework/cunha001/index.html).] The kidneyinherently has little stroma to interfere with implantedtissues and a rich vasculature that ensures the growthand survival of the implants. These characteristics makethe kidney a suitable environment for the rescue of im-portant tissues. To take advantage of stromal condi-tional knock-out mice, renal capsule allografting, aswell as an intact immune system, we have successfullyperformed tissue rescues of prostate from Tgfbr2fspKO

mice in syngenic male C57/Bl6 mice enabling survival ofthe rescued prostate for up to a year from mice thatwould otherwise die by 6–7 weeks of age.

Tissue recombination grafting provides another al-ternative to the creation of transgenic mice for thein vivo examination of gene function and interrogationof signal transduction pathways. For instance, to studyprostate stromal–epithelial interactions, epithelial cellsor intact prostatic organoids (in which the stroma hasbeen removed by digestion with collagenase and DNa-se-I) can be recombined with stromal cells within amatrix of rat-tail collagen (Hayward et al., 1999). Thesegrafts can be implanted immediately or incubated over-night and implanted the next day under the renal cap-sule of host mice (as described above). Recently, aUGM cell line (UGSM-2) has been isolated from anE16 mouse UGS. These cells participate in prostateglandular morphogenesis when recombined with UGEand may be a useful tool for gain- and loss-of-functionstudies in prostate tissue recombination models (Shawet al., 2006; Shaw et al., 2008, this issue). Compartment-specific gene modulation is achievable through use oflentiviruses, which can infect both proliferative and

non-proliferative cells (Naldini et al., 1996), to express agene of interest or to knock-down a gene of interest byRNAi before the creation of the graft (Williams et al.,2005; Ao et al., 2006, 2007; He et al., 2007). Ad-Cre orcell-permeable Cre may also be used in culture or whenassembling the graft to knock-out the expression of afloxed gene in situ.

Concluding remarks

While use of conditional promoter systems to createtransgenic knock-in and knock-out mouse models havebeen invaluable, the technology available to date has yetto overcome long-standing challenges whose solution isessential to the molecular dissection of stromal tissues(Schmidt-Supprian and Rajewsky, 2007). Currentlyavailable promoter-regulated Cre’s still target thestroma of multiple tissues often with mosaicism. Thisadds further variability to stromal compartments thatare frequently heterogeneous in nature within normaltissues and between different tissues and organs. More-over, currently available promoter-regulated Cre’s aregenerally not specific enough to hit just the desiredstromal compartment or cell-lineage within a singlespecific tissue. The ability to dissect functionally thestromal microenvironment during development or tostudy carcinogenesis is a balance of the stromal spec-ificity of the targeting mechanism and inherent stromalheterogeneity. One solution can be to develop condi-tional knock-out mice using combinatorial promoters,which will specifically restrict temporal (developmental)and spatial (compartment or cell-lineage specific) expres-sion with higher fidelity than endogenous promoters.

In summary, the development and use of conditionaltransgenic mouse models has increased our understand-ing of the epithelial and stromal compartments of tis-sues including the prostate. Refinement of currentlyavailable transgenic technology as well as further elu-cidation and delineation of cell and tissue-specific differ-entiation markers in stromal tissues will improve ourability to create increasingly informative mouse modelsutilizing targeted gene expression to and within specificstromal compartments.

Acknowledgments The authors would like to thank Simon Hay-ward for critical review of the manuscript as well as helpful com-ments and suggestions. The work was supported by the NIHthrough CA108646 and CA126505, and the DOD through DAMD17-02-1-0063 and W81XWH-04-1-0046.

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