Lawrence Berkeley National Laboratory, 1 Cyclotron Road, MS 83-101, Berkeley, CA 94720, USA
Correspondence (emails: mjbissell{at}lbl.gov; klschmeichel{at}lbl.gov)
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Summary |
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Key words: Human epithelial cells, Three dimensional, Organotypic models, Tissue-specific signaling
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Introduction |
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Because of the ethical, technical and financial constraints inherent in
research on human cells and tissues, the demand for models that faithfully
parallel human form and function considerably outweighs the supply. We and
others asserted more than two decades ago that development of physiologically
relevant models of both rodent and human origin should recognize that organs
and tissues function in a 3D environment
(Elsdale and Bard, 1972;
Hay and Dodson, 1973
;
Emerman and Pitelka, 1977
;
Bissell, 1981
;
Ingber and Folkman, 1989
).
Further, that in the final analysis, the organ itself is the unit of function
(Bissell and Hall, 1987
). We
now know that exposure of cells to the spatial constraints imposed by a 3D
milieu determines how cells perceive and interpret biochemical cues from the
surrounding microenvironment [e.g. the extracellular matrix, growth factors
and neighboring cells (for reviews, see
Roskelley et al., 1995
;
Bissell et al., 2002
;
Cunha et al., 2002
;
Ingber, 2002
;
Radisky et al., 2002
)].
Furthermore, it is in this biophysical and biochemical context that cells
display bona fide tissue and organ specificity.
Here, we describe studies of epithelial-cell-based systems that demonstrate the importance of developing and utilizing 3D human organotypic models to understand the molecular and cellular signaling events underlying human organ biology (Fig. 1). In their most simplistic form, these models comprise homogeneous epithelial cell populations that are cultured within 3D basement-membrane-like matrices. These relatively simple `monotypic' cell models have progressively evolved into 3D co-culture models containing multiple cell types, which approximate organ structure and function in vitro and enable systematic analyses of the molecular contributions of multiple cell types. Finally, we go on to explore how human 3D culture models are being coupled to existing technologies in the mouse to generate models in vivo that could elucidate the fundamental influence of stromal-epithelial interactions in normal organ function as well as those that perturb organ homeostasis and lead to disease. As a result of these advancements, we are equipped with a hierarchy of related models that appreciate the importance of 3D environments but vary with respect to cellular complexity. By using this collection of experimental models interchangeably, we can test molecular markers and targets in models of defined at the molecular level with increasing physiological relevance (culminating in vivo); conversely, results in vivo can be translated into less complex models that are more suitable for diagnostic and therapeutic development.
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Monotypic 3D cell culture assays |
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A number of cell models have been coupled with appropriate 3D matrices and
show fruitful results in recapitulating tissue functions in 3D. Extensive
studies have been reported for liver, salivary gland, vasculature, bone, lung,
skin, intestine, kidney and mammary and thyroid glands (see
Table 1 and references
therein). Other cells, such as MDCK
(O'Brien et al., 2001;
Troxell et al., 2001
) and
fibroblasts (Harkin and Hay,
1996
; Cukierman et al.,
2001
), have also been monitored in 3D contexts and have provided
valuable insight into the basic molecular mechanisms of polarity, branching
morphogenesis, adhesion and cell migration (reviewed in
Cukierman et al., 2002
;
O'Brien et al., 2002
;
Walpita and Hay, 2002
).
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In some cases, however, the relationship between these models in culture and a counterpart in vivo is unclear. We contend that, in order to be useful as a translational tool for the study of human disease progression, 3D organotypic models must be developed that are true to human form and function. In the following sections, we will describe the development of 3D culture models in human breast (and, briefly, in skin) that demonstrate how appropriate choice of cell source and ECM substrata can enable the establishment of physiologically relevant assay systems. By virtue of their resemblance to organ structure and function in vivo, these models facilitate meaningful dissection of the molecular mechanisms involved in the regulation of tissue specificity.
Monotypic 3D cultures for modeling mammary gland and epithelial
signaling
A large body of work performed in mammary epithelial cells from mice
demonstrates the central importance of 3D cell-microenvironment interactions
in promoting a differentiated cellular response
(Lin and Bissell, 1993;
Roskelley et al., 1995
;
Boudreau and Bissell, 1998
).
Mammary cells embedded in lrBM adopt a spherical, polarized structure that
resembles the normal mammary alveolus (or acinus) and that is capable of
mammary-gland-specific function (e.g., producing milk in response to
lactogenic hormones) (Barcellos-Hoff et
al., 1989
) (reviewed in
Stoker et al., 1990
). Human
luminal epithelial cells, both primary and immortalized, respond to ECM in
much the same way as their mouse counterparts by forming acini in 3D
(Fig. 2)
(Petersen et al., 1992
;
Howlett et al., 1995
;
Weaver et al., 1997
). [The
same cells grown in an interstitial ECM, such as collagen I, show altered
integrins and abnormal polarity and organization
(Howlett et al., 1995
;
Gudjonsson et al., 2002a
;
Weaver et al., 2002
), thereby
underscoring the importance of matching cell types with appropriate
substrata.] Human breast tumor cells fail to show a differentiated phenotype
in 3D lrBM, but instead form cellular masses that are disorganized and apolar
(Petersen et al., 1992
).
Collectively, these studies demonstrate that human mammary epithelia respond
to structural and biochemical cues provided by the ECM and that these cell-ECM
interactions are sufficient to reveal innate cellular phenotypes.
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3D organotypic cultures are amenable to a variety of experimental manipulations and have been effectively used to re-examine molecular pathways previously characterized by conventional culture methodologies as well as to elucidate novel signaling pathways. Recent examples are described below and are summarized in Fig. 2.
Coupled signaling mechanisms and reversion
When used in conjunction with well-defined human mammary cell cultures,
monotypic 3D assays have proven useful for understanding how altered cell-ECM
communication regulates breast tumor progression. One such cell source is the
HMT-3522 human breast tumor progression series, which comprises a continuum of
cell populations that arise from a common precursor but range in phenotype
from non-malignant (S1) to tumorigenic (T4-2)
(Briand et al., 1987;
Nielsen et al., 1994
;
Briand et al., 1996
).
Dissection of the molecular differences between these cells revealed that
surface expression of the ECM receptor ß1 integrin is dramatically
upregulated in T4-2 tumor cells in comparison with their non-malignant
counterparts. When treated in 3D cultures with antibodies that block ß1
integrin function, T4-2 cells dramatically reorganize: cell colonies become
phenotypically reverted, assuming a polarized and growth-arrested status
comparable to that observed of non-malignant S1 cells
(Fig. 2) (Weaver et al., 1997
).
Reduced ß1 integrin signaling results in downregulation of endogenous
ß1 integrins as well as in reduced signaling and levels of the epidermal
growth factor receptor (EGFR) (Wang et
al., 1998
). In a reciprocal fashion, neutralizing the activity of
EGFR effectively normalizes ß1 integrin signaling and levels
(Fig. 2). Such ß1 integrin
and EGFR reciprocal cross-modulation is apparently dependent upon a 3D context
as neutralizing antibodies do not elicit reciprocal cross-modulation in cells
cultured as 2D monolayers (Wang et al.,
1998
).
To determine the extent to which adhesion and growth factor receptor
signaling is coupled in more aggressive tumors, Wang et al. recently assayed a
series of metastatic human breast cancer cell lines for sensitivity to ß1
integrin and EGFR cross-talk inhibitors in 3D lrBM cultures
(Wang et al., 2002). Unlike
results from the tumorigenic, but non-metastatic, T4-2 cells, single
inhibitors induce only partial phenotypic reversion in aggressive carcinoma
cells (Fig. 2). Instead,
specific pairs of inhibitors, applied in tandem, are required to revert the
malignant phenotype or cause apoptosis. Therefore, as signaling pathways
become increasingly disconnected, they require intervention at multiple sites
to elicit reversion and/or apoptosis.
Collectively, these studies show that recapitulation of phenotypically normal tissue in 3D lrBM assays correlates with the ability of adhesion and growth factor receptor signaling pathways to engage in reciprocal cross-modulation. Moreover, the intracellular signaling pathways directing cell polarity and proliferation in human mammary epithelial tissues are orchestrated in profoundly different ways, depending on whether cells are cultured in a 2D or 3D context. These studies also constitute proof that 3D assays of phenotypic reversion can be exploited further to characterize potential modulators of the malignant phenotype in breast.
Tumor suppressors and oncogenes
3D lrBM assays can be used to search for oncogenes and tumor suppressors as
well as to understand their mechanisms of action. For example, Howlett et al.
examined the extent to which restoration of Nm23-H1, a metastasis-suppressor
gene (Leone et al., 1993),
could restore `normal' cell morphology
(Howlett et al., 1994
). They
transfected a metastatic breast carcinoma cell line MDA-MB-435 with an Nm23-H1
transgene and assayed the resulting transgenic cells 3D lrBM cultures. They
found that cells overexpressing Nm23-H1 formed organized acinus-like spheres
with appropriately polarized basal and apical surfaces
(Fig. 2) and thus provided
evidence that suppressive effects of Nm23-H1 might be due to its role in
growth inhibition and differentiation in response to cues from the ECM.
Spancake et al. examined the effects of downmodulating the retinoblastoma (RB)
tumor-suppressor pathway in human mammary epithelial cells. Whereas loss of RB
function did not affect polarity of these epithelial cells in 3D
(Spancake et al., 1999
), the
3D acini were not growth arrested and failed to display several markers of
differentiation found in primary cells, thereby demonstrating that RB function
plays a role in mammary cell differentiation.
AZU-1 is a gene product that was isolated by comparative gene expression
analysis of premalignant and malignant cells of the HMT-3522 series
(Chen et al., 2000).
AZU-1 [also called TACC2
(Still et al., 1999
)] mRNA is
significantly downregulated in a variety of human breast tumor cells, which is
consistent with it having a tumor suppressor role in breast tissue.
Normalizing the expression of AZU-1 in T4-2 tumor cells causes phenotypic
reversion of the cells, as revealed in 3D lrBM assays. These findings, in
combination with in vivo tumorigenicity assays, provides experimental evidence
that AZU-1 is a novel breast tumor suppressor. Interestingly, AZU-1 levels,
which are very low in T4-2 cells, become normalized in tumor cells reverted by
EGFR or ß1 integrin inhibition, which suggests that AZU-1 expression is
also sensitive to cues from the microenvironment
(Chen et al., 2000
).
More recently, the non-integrin cell surface ECM receptor dystroglycan (DG)
was also shown to display a tumor-suppressive function in T4-2 cells
(Muschler et al., 2002).
Re-expression of DG in tumor cells lacking
-DG expression but
expressing E-cadherin produced profound repolarization of cells in the 3D lrBM
assay. This finding suggests that, at the cell surface, normal cellular
function might be a result of a competitive balance that is achieved by
signaling through integrins, growth factors and dystroglycan.
Novel aspects of the function of the oncogene ErbB2 in tumor progression
have been revealed in 3D human mammary epithelial cell cultures as well. ErbB2
is particularly interesting as a potential oncogene in the breast because its
overexpression correlates with a poor clinical prognosis (reviewed in
Eccles, 2001;
Yarden, 2001
). Human MCF10A
mammary epithelial cells were recently engineered to express conditionally
activated ErbB2 and analyzed in monotypic 3D cultures
(Fig. 2)
(Muthuswamy et al., 2001
).
When ErbB2 receptor, but not ErbB1, is activated in mature 3D acini, the
MCF10A cells lose their polarized organization and develop structures
consisting of multiple acinar-like units with filled lumina. These structures
do not display any invasive properties and thus represent a reasonable model
for ductal carcinoma in situ (DCIS). This finding also raised the possibility
that excessive signaling through ErbB2 in 3D cultures is sufficient to induce
growth and to protect cells from apoptosis within the luminal space
(Huang et al., 1999
;
Muthuswamy et al., 2001
) (see
also discussion below).
Sensitivity and resistance to cell death
More than two decades ago, Hall et al. showed that, when murine mammary
cells are sandwiched between two layers of ECM, they form a lumen
(Hall et al., 1982). Since
then, 3D monotypic cultures have been used to explore the basic developmental
pathways of the mammary gland, including lumen formation. The work of Frisch
and colleagues indicated that adhesion to any ECM molecule is a survival cue
and that loss of adhesion hastens a cell's demise (anoikis)
(Frisch and Francis, 1994
).
Boudreau et al. showed that adhesion to an inappropriate ECM ligand, at least
in the case of epithelial cells, only delays cell death temporarily
(Boudreau et al., 1995
) and
that adhesion to relevant substrata, such as lrBM or laminin, is necessary to
maintain long-term survival. Once the cells lose contact with BM, caspases are
induced and the cells apoptose. Coucouvanis and Martin showed subsequently in
developing mouse embryos that, whereas contact with BM protects the outer cell
layer, the inner cell mass cavity is carved by apoptosis of cells that had no
contact with BM (Coucouvanis and Martin,
1995
). Others have since shown that cavitation of the lumen of the
mammary gland is also mediated by induction of apoptosis
(Blatchford et al., 1999
) (see
also below).
Several recent reports using 3D human mammary epithelial cell models have
provided important insights into the molecules and pathways involved in the
apoptotic events leading to lumen formation. For example, Huang et al. showed
that biliary glycoprotein (BGP, also known as CEACAM1 or CD66a), a
transmembrane protein expressed on the luminal surface of mammary epithelia,
is required for lumen formation in MCF10A-derived acini cultured in 3D lrBM
(Huang et al., 1999).
Re-expression of a short isoform of BGP in MCF7 breast carcinoma cells (that
is, cells that lack BGP and fail to form lumena in 3D lrBM assays) results in
cells that form morphologically normal mammary acini with properly formed
central lumena in 3D cultures (Kirshner et
al., 2003
). This model revealed that BGP adopts an apical
localization during morphogenesis and influences lumen formation by initiation
of apoptotic pathways.
Two recent studies by Muthuswamy et al. and Debnath and colleagues show
that lumen formation in the MCF10A model is not mediated solely by the action
of pro-apoptotic signals (Muthuswamy et
al., 2001; Debnath et al.,
2002
). Rather growth control signals and increased apoptotic
signaling cooperate to direct lumen formation in this 3D monotypic model.
Because chronic activation of ErbB2 is sufficient to cause accumulation of
colonies with cell-filled lumina, ErbB2 oncogenic signaling probably exerts
multiple biological effects, coordinating signals that affect both
proliferation and apoptosis during cavitation
(Debnath et al., 2002
). These
studies provide a compelling example of how the study of developmental
processes (such as lumen formation) can benefit from the experimental
accessibility of these simple tissue-specific models to yield physiologically
relevant information at the molecular level.
The HMT-3522 culture model was recently employed to address mechanisms of
resistance to chemotherapeutic-agent-induced apoptosis in vivo
(Weaver et al., 2002; see
also Yamada and Clark, 2002
).
Formation of 3D polarized structures confers protection against apoptosis in
both non-malignant and malignant mammary epithelial cells. Destabilizing the
polarity of these structures by disrupting ß4 integrin ligation, and thus
perturbing hemidesmosome organization, allows induction of apoptosis. Loss of
ß4 integrin results in the inactivation of NF
B, a known positive
modulator of expression and stability of apoptosis regulators. This topic is
of critical relevance to considerations of apoptotic drug resistance and tumor
dormancy. Metastasized tumor cells, either as single cells or clusters, may
resist death induced by chemotherapeutic agents when the microenvironment and
spatial information at the secondary site is conducive to the establishment of
cell polarity and survival.
The functional significance of nuclear structure
The status of nuclear organization is an important indicator of tissue
homeostasis and differentiation in vivo
(Lelievre et al., 2000;
Nickerson, 2001
). In 3D
cultures, the nuclear structure of HMT-3522-S1 cells differs radically from
that of the same cells cultured in a monolayer
(Lelievre et al., 1998
).
Furthermore, a series of structural changes are evident within the nucleus
throughout acinar morphogenesis in 3D. The observed structural modifications
are apparently coupled to S1 cell function, as targeted disruption of the
nuclear structure in fully differentiated S1 cells causes upregulation of
matrix metalloproteinase activity, an event that ultimately alters the quality
of the underlying basement membrane
(Lelievre et al., 1998
).
Collectively these studies point to a dynamic and reciprocal functional
connection between nuclear structure and cell function that is dependent upon
the presence of an appropriate 3D context. Much remains to be understood about
how communication between the BM, the 3D structure and the nucleus is
established. However, the idea that understanding these connections requires
3D models is now being more widely championed. Commenting on recent papers by
Debnath et al. and Weaver et al., Jacks and Weinberg conclude:
"Suddenly, the study of cancer cells in two dimensions seems quaint, if
not archaic" (Debnath et al.,
2002
; Weaver et al.,
2002
; Jacks and Weinberg,
2002
).
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Modeling organs in vitro: organotypic co-cultures |
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The co-culture paradigm: human skin
3D organotypic co-culturing methodologies have been particularly successful
in epidermal biology. A cultured version of a `skin equivalent' has been
achieved through culturing of keratinocytes either on de-epidermized dermis
(Regnier et al., 1981;
Watt, 1988
;
Fartasch and Ponec, 1994
) or
on collagen gels embedded with dermal fibroblasts
(Bell et al., 1981
;
Regnier et al., 1981
;
Asselineau and Prunieras, 1984
;
Asselineau et al., 1985
;
McCance et al., 1988
;
Watt, 1988
;
Coulomb et al., 1989
;
Fartasch and Ponec, 1994
).
Such co-cultures give rise to stratified epithelium that displays many of the
morphological and functional features of an epidermis in vivo
(Bell et al., 1981
;
Kopan et al., 1987
;
Watt, 1988
;
Kopan and Fuchs, 1989
;
Hertle et al., 1991
;
Parenteau et al., 1991
;
Fusenig, 1994
;
Smola et al., 1998
).
Differentiated human skin equivalents are produced in co-cultures
containing fibroblasts of either human or mouse origin
(Choi and Fuchs, 1990;
Turksen et al., 1991
;
Kaur and Carter, 1992
). Given
this inherent compatibility, which probably reflects a similarity in the
synthesis of paracrine factors between species, one can supplement skin
co-cultures with fibroblasts derived from genetically engineered mice. An
elegant example of such a substituted culture allowed examination of the role
of AP-1 transcription factor subunits c-jun and junB in skin homeostasis
(Fig. 3)
(Szabowski et al., 2000
;
Angel and Szabowski, 2002
).
Unlike wild-type murine fibroblasts, the presence of c-jun-deficient or
junB-deficient fibroblasts had dramatic and distinct effects on keratinocyte
proliferation and differentiation when included in skin co-cultures. These
studies elucidated a double paracrine mechanism in which keratinocytes produce
Il-1, which, in turn, induces the expression of keratinocyte growth factor
(KGF) and granulocyte macrophage colony stimulating factor (GM-CSF) in dermal
fibroblasts through AP-1 activation
(Maas-Szabowski et al., 2000
;
Szabowski et al., 2000
;
Maas-Szabowski et al.,
2001
).
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When used in combination with skin tumor progression models, 3D co-cultures
also effectively distinguish between non-malignant and malignant phenotypes.
Such strategies were used to show that MMP1, a matrix metalloproteinase
implicated in tumor induction and progression, is upregulated not only in more
aggressive tumor types but also in the co-cultured fibroblasts themselves
(Fusenig and Boukamp, 1998).
Other studies have established that embryonic stem (ES) cells from normal
animals can produce well-differentiated epidermis in 3D co-cultures
(Bagutti et al., 1996
). Taking
advantage of this compatibility, Bagutti et al. recently demonstrated that
ß1-integrin-null stem cells respond and differentiate in epidermal skin
cultures only in the presence of excess stromal factors and that loss of
ß1 integrin decreases the sensitivity of ES cells to soluble factors that
induce differentiation (Bagutti et al.,
2001
).
Given the development of these tractable models of human skin that display
an unquestionable resemblance to skin in vivo, it is not surprising that these
models are being productively utilized in pharmacotoxicological studies
(Gay et al., 1992) and in skin
grafting procedures (Boyce and Warden,
2002
). Furthermore, they provide critical guidance for modeling
the complexities of other human organ systems, such as the breast, in
culture.
Modeling breast complexity in culture
The unit morphology of a mammary duct is a double-layered structure in
which a continuous sheet of polarized epithelium is surrounded by a layer of
myoepithelial cells. Because of their contractile nature, myoepithelial cells
have generally been recognized for their function in the extrusion of secreted
milk from mammary gland during lactation. However, accumulating evidence
indicates that myoepithelial-luminal epithelial cell interactions contribute
to homeostasis within the mammary gland and that disruption of this
interaction might be an important step in tumor progression
(Zou et al., 1994;
Sternlicht et al., 1997
;
Man, 2002
).
Recently, several studies performed in 3D organotypic co-culture models
have examined the role of myoepithelia in organizing and maintaining normal
gland structure and function. Runswick and colleagues mixed purified human
luminal epithelial cells and myoepithelial cells and incubated them in a 3D
rotary culture environment (Runswick et
al., 2001). Under these conditions, double-layered structures
formed, containing a central core of polarized luminal epithelial cells
surrounded by a layer of myoepithelial cells. Perturbation of
myoepithelium-specific desmosomal cadherins disrupted basal positioning of
myoepithelial cells (Runswick et al.,
2001
), thereby demonstrating that physical associations between
myoepithelial and luminal epithelial cells are important for the establishment
of higher-order organ structure in the mammary gland.
The interdependence of luminal and myoepithelial cells has also been
analyzed in 3D ECM cultures in which purified primary luminal epithelial cells
were combined within 3D collagen I gels in the presence or absence of purified
myoepithelial cells (Gudjonsson et al.,
2002a). As expected from previous studies of human mammary
epithelial cells in 3D collagen I cultures
(Howlett et al., 1995
;
Lelievre et al., 1998
), the
primary luminal epithelial cells alone fail to show appropriate polarity in 3D
collagen I. However, collagen-based co-cultures containing both luminal
epithelial and myoepithelial cells show polarized, bilayered organization
(Fig. 4). Normal myoepithelial
cells direct luminal epithelial cell polarity by synthesizing laminin 1;
tumor-derived myoepithelial cells, expressing no or low levels of laminin 1,
fail to yield double-layered organotypic structures in 3D collagen co-cultures
(Gudjonsson et al., 2002a
).
Thus, the basal positioning of myoepithelial cells is not only well-suited for
contractile events that occur during lactation (a known function of
myoepithelial cells) but also provides important spatially restricted
biochemical cues that drive cell polarity and normal function in the breast
(see Bissell and Bilder,
2003
).
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Indeed, it is now conceivable that the human breast could be reasonably
recapitulated in vitro by systematic pairing of different cell types in 3D
culture. In a recent report, Hass and Kratz reported the results of
co-culturing primary human mammary epithelial cells and adipocytes derived
from the same patient (Huss and Kratz,
2001). In these cultures, differentiated epithelial structures are
embedded in clusters of adipocytes in patterns reminiscent of the human breast
in vivo. Whether these structures reflect the ability of adipocytes to
contribute to a BM and whether there are other functions provided by the fat
cells remain to be determined. Additional modeling work, perhaps incorporating
stem cell populations as described above for skin or immortalized progenitor
cells of the breast (Gudjonsson et al.,
2002b
), will be required if we are to achieve the full complement
of mammary gland components in 3D culture.
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Stromal-epithelial interactions in 3D in vivo |
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One compelling demonstration of the stromal control of epithelial behavior
comes from Olumi and colleagues, who developed a model in which non-malignant
prostatic epithelial cells (normal or SV40-immortalized) were mixed in
collagen gels with prostate-derived fibroblasts from normal epithelial
organoids or cultures of carcinoma-associated cells. These 3D cultures were
then transplanted beneath the renal capsule of athymic mice, and epithelial
outgrowth was monitored (Olumi et al.,
1999). Neither epithelial cells nor fibroblasts, alone, promoted
tumor formation on their own. However, xenografts that included
carcinoma-associated fibroblasts (CAFs) along with SV-40-immortalized
epithelial cells shows a dramatic tumorigenic response. These findings
demonstrate in an in vivo setting that the altered signaling capacity of the
CAFs is sufficient to catalyze tumor progression in a cell type that displays
a mildly altered genotype.
Parmar et al. have recently used a similar renal grafting approach to
address the role of mammary stromal fibroblasts in mammary gland function and
development (Parmar et al.,
2002). They mixed normal human mammary epithelial cells, prepared
as organoids, with mammary fibroblasts and grafted them into renal capsules of
nude mice. These transplants show a robust elaboration of a mammary structure,
which does not occur in the absence of mammary stroma and appears to be
responsive to hormonal stimulation from estrogen and progesterone (e.g., they
can be induced to produce milk when analyzed in pregnant animals)
(Parmar et al., 2002
).
Human xenograft and tissue transplant models are thus powerful tools for
analyzing the complexities of organ function, especially when results can be
reciprocally tested and scrutinized in simpler 3D culture models. However,
opinions differ with respect to the optimum site of tissue transplantation
within the animal and its preparation. Does the outgrowth of human mammary
epithelium in the kidney, for example, truly represent normal mammary events
or does the mouse fat pad provide a more relevant environment for outgrowth?
Indeed, it has long been known that mouse mammary epithelial cells display
differential developmental responses depending upon their site of delivery
(see Miller et al., 1981;
Neville et al., 1998
). A
recent study revealed that human mammary epithelial cells harboring three
cancer-predisposing genetic alterations have differential tumorigenic
responses in nude mice, the most extreme response being associated with
transplantation into cleared mammary fat pad
(Elenbaas et al., 2001
).
Considering the range of responses from both `normal' and tumorigenic cells
within a given animal, perhaps we should also ask whether the rodent fat pad
is of sufficient relevance to reconstitute a human cell behavior that is true
to the human form or whether we should be striving to humanized mouse mammary
models?
Developing a `humanized' mammary gland in the mouse fat pad
In very general terms, the mouse and human mammary glands share a
reasonable level of similarity but also some differences
(Fig. 1) (Ronnov-Jessen et al., 1996).
Because the mouse mammary gland has high levels of adipose stroma, it is
reasonable to suggest that the environment of the mouse mammary fat pad in
mice is not entirely equivalent to human breast
(Neville et al., 1998
).
Cleared fat pads, commonly used in transplant studies, also include several
other cell types, such as fibroblasts, endothelial cells and cells of the
immune system, all of which could influence mammogenesis. Moreover, techniques
routinely used to prepare orthotopic sites, such as irradiation, may in fact
induce profound and lasting stromal effects by themselves
(Barcellos-Hoff and Ravani,
2000
; Barcellos-Hoff,
2001
). Furthermore, human mammary epithelial cells injected into
cleared fat pads do not elaborate ductal structures
(Sheffield, 1988
), which
emphasizes the apparent incompatibility between human and mouse mammary
organs.
To study breast homeostasis with ultimate physiological relevance, we must
model not just tissues but entire organs in vivo
(Bissell and Hall, 1987;
Bissell and Radisky, 2001
).
One future goal would be to develop `humanized' mammary glands in rodents by
replacing mouse mammary gland components with their human counterparts and
reconstructing an organ that is comparable to the human gland in its
architecture and organization (Fig.
1). As daunting as this task seems, progress is currently being
made to craft such a `humanized' mammary gland model in mouse. Human stromal
fibroblasts grafted into the fat pads of nude mice support elaboration of
transplanted human mammary epithelial cells into an infiltrating ductal tree
that is highly differentiated and responsive to lactogenic cues (C.
Kuperwasser and R. A. Weinberg, personal communication). Establishment of such
a `humanized' animal paves the way for the systematic inclusion of genetically
modified epithelial or stromal cells and thereby provides a model for
controlled analysis of specific molecules and pathways in an in vivo context.
Furthermore, the `humanized' mouse could also be important in the development
of strategies for reconstructing human breast after surgical intervention
(Huss and Kratz, 2001
).
Future versions of `humanized' mice could incorporate human mammary
progenitor cells in this type of transplant model. The existence of mammary
epithelial stem cells has been the subject of much debate, but recent evidence
demonstrates the existence of precursor cells within the luminal epithelial
pool (Smith, 1996;
Stingl et al., 1998
;
Smalley et al., 1999
).
Recently, Gudjonnson et al. have isolated and immortalized a human mammary
progenitor cell that gives rise to structures that resemble terminal ductal
lobular units (TDLU) when implanted either in Matrigel or orthotopically into
nude mice (Gudjonsson et al.,
2002b
). Thus it seems plausible that introduction of human
progenitor cells in the mouse mammary gland may be a useful strategy for
generating chimeric animals with extensively humanized mammary organs.
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Concluding remarks |
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Acknowledgments |
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References |
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