Mammary collective cell migration involves transient
loss of epithelial features and individual cell migration
within the epithelium
Andrew J. Ewald1,2,*, Robert J. Huebner2, Hildur Palsdottir3, Jessie K. Lee3, Melissa J. Perez3,
Danielle M. Jorgens3, Andrew N. Tauscher3, Kevin J. Cheung2, Zena Werb1,`and Manfred Auer3,`
1Department of Anatomy, University of California-San Francisco, 513 Parnassus Avenue, Box 0452, San Francisco, CA 94143, USA
2Departments of Cell Biology and Oncology, Center for Cell Dynamics, Johns Hopkins School of Medicine, 855 N. Wolfe Street, 452 Rangos Building,
Baltimore, MD 21205, USA
3Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, MS Donner, Berkeley, CA 94720, USA
*Author for correspondence (email@example.com)
`These authors contributed equally to this work
Accepted 31 January 2012
Journal of Cell Science 125, 2638–2654
? 2012. Published by The Company of Biologists Ltd
Normal mammary morphogenesis involves transitions between simple and multilayered epithelial organizations. We used electron
microscopy and molecular markers to determine whether intercellular junctions and apico-basal polarity were maintained in the
multilayered epithelium. We found that multilayered elongating ducts had polarized apical and basal tissue surfaces both in three-
dimensional culture and in vivo. However, individual cells were only polarized on surfaces in contact with the lumen or extracellular
matrix. The basolateral marker scribble and the apical marker atypical protein kinase C zeta localized to all interior cell membranes,
whereas PAR3 displayed a cytoplasmic localization, suggesting that the apico-basal polarity was incomplete. Despite membrane
localization of E-cadherinand b-catenin, we didnotobserve a defined zonulaadherens connecting interior cells. Instead, interior cells were
connected through desmosomes and exhibited complex interdigitating membrane protrusions. Single-cell labeling revealed that individual
cells were both protrusive and migratory within the epithelial multilayer. Inhibition of Rho kinase (ROCK) further reduced intercellular
adhesion on apical and lateral surfaces but did not disrupt basal tissue organization. Following morphogenesis, segregated membrane
domains were re-established and junctional complexes re-formed. We observed similar epithelial organization during mammary
morphogenesis in organotypic culture and in vivo. We conclude that mammary epithelial morphogenesis involves a reversible, spatially
limited, reduction in polarity and intercellular junctions and active individualistic cell migration. Our data suggest that reductions in
polarity and adhesion during breast cancer progression might reflect partial recapitulation of a normal developmental program.
Key words: Epithelial morphogenesis, Apico-basal polarity, Mammary gland, Breast cancer
Simple epithelial tissues are composed of a single layer of cells
connected to each other through intercellular anchoring junctions,
including tight junctions, adherens junctions and desmosomes.
Epithelial morphogenesis therefore presents a conceptual puzzle, as
adhesive junctions and strong apico-basal cell polarity are difficult to
reconcile with models of motility derived from the study of isolated
fibroblastic cells migrating over flat surfaces (Lauffenburger and
Horwitz, 1996; Nelson, 2009). A major barrier to the understanding
of mammalian epithelial morphogenesis is that the development
occurs deep inside the animal. The mammary gland poses a
particular challenge because the developing ductal network is
embedded within an adipocyte-rich stroma that scatters light and
limits high-resolution imaging. Consequently, mammary ductal
morphogenesis has not been directly observed in vivo and the basic
events are inferred from analysis of fixed specimens.
multilayered epithelial placode during embryonic development
(Hogg et al., 1983). The epithelium internalizes and elongates
into the embryonic mesenchyme as a solid, multilayered cord of
cells from embryonic days 12–15. Although the mammary cells
make extensive cell–cell contacts, they lack many features of
mature epithelium, such as tight and adherens junctions (Hogg
et al., 1983). During embryonic days 16–20 the epithelium
architecture with a central lumen and mature junctional
complexes (Hogg et al., 1983). This rudimentary ductal
network is then essentially inactive until the increase in steroid
hormone levels at the onset of puberty. The majority of
mammary branching morphogenesis occurs during puberty
(Fig. 1A). At the end of embryonic development and at the
end of puberty, mammary ducts consist of a highly polarized
luminal epithelial cell layer surrounded by myoepithelial cells
(Fig. 1B,C). However, ductal elongation during puberty is
accomplished by a specialized structure known as the terminal
end bud (TEB; Fig. 1D). The TEB contains many luminal
epithelial cell layers (Williams and Daniel, 1983), and these cells
display incomplete apico-basal polarity as determined by light
microscopy (Hinck and Silberstein, 2005; Mailleux et al., 2007;
Ewald et al., 2008).
Journal of Cell Science
We developed an ex vivo culture system to enable direct
observation of the cell dynamics driving mammary epithelial
morphogenesis (Simian et al., 2001; Wiseman et al., 2003;
Sternlicht et al., 2005; Fata et al., 2007; Ewald et al., 2008). In the
‘organoid’ assay we isolate primary mammary epithelial ducts and
embed them inside gels of extracellular matrix proteins. Epithelial
organoids in three-dimensional (3D) culture initiate, elongate and
bifurcate new ducts in a growth-factor-dependent fashion (Fata
et al., 2007; Ewald et al., 2008). We used immunofluorescence to
verify that the organization and polarity of the epithelium during
morphogenesis in 3D culture is highly similar to that in the TEB in
vivo (Ewald et al., 2008). We next developed microscopy
techniques to visualize the cell dynamics driving mammary
morphogenesis in 3D culture (Ewald, 2010). In time-lapse movies
we observed a proliferation-dependent transition from a simple
to a multilayered epithelial organization and dynamic cell
rearrangements within the epithelial layer (Ewald et al., 2008).
We did not observe any actin- or membrane-based protrusions
extending into the extracellular matrix (ECM). Luminal epithelial
buds initiate and elongate as a multilayered epithelium, with active
cell rearrangements and without persistent leader cells. As
elongation ceases, the epithelium returns to a simple bilayered
organization (Ewald et al., 2008). We conclude that the mammary
epitheliumduring morphogenesis in 3D culture is both
multilayered and highly dynamic at the cellular level.
Studies in kidney and salivary gland have revealed that
epithelial cells exhibit high levels of motility and extensive cell
rearrangements during morphogenesis (Larsen et al., 2006; Chi
et al., 2009). However, those organs accomplish branching
morphogenesis during embryonic development and these cell
dynamics are essentially absent by birth (Larsen et al., 2006).
Similarly, at the end of puberty all of the TEBs disappear and
the mammary ductal network again has a simple bilayered
organization and extensive intercellular junctions. However,
mammary epithelial fragments isolated from mice of any age
will reform TEBs and re-establish a complete ductal epithelial
network when transplanted into a cleared mammary fat pad
in a host mouse (Daniel et al., 1968). Consistent with these in
vivo observations, mammary ducts isolated from embryonic,
adolescent and mature mice all branch in 3D organotypic culture
and display fundamentally similar cellular dynamics. Mammary
epithelial ducts elongate through the action of a multilayered
TEB and the epithelium retains the potential to reform TEBs
throughout the lifespan. We therefore became specifically
interested in determining the extent to which intercellular
adhesive junctions and apicobasal polarity are maintained in
this transient epithelial multilayer.
The major adhesive junctions in epithelial cells are the tight
junctions, adherens junctions and desmosomes. Tight junctions
are composed of claudins and occludins and they serve both to
control paracellular permeability and to partition the plasma
membrane into apical and basolateral domains (Schneeberger and
Lynch, 2004). Adherens junctions directly connect cells through
classical cadherins and then link cell contacts to the actin
Wheelock, 2005). Desmosomes link cells through desmosomal
cadherins and connect these cell contacts to the intermediate
filament cytoskeleton (Getsios et al., 2004). In addition to the
structural polarity imposed by the localization of the adhesive
junctions, epithelial cells also receive molecular polarity
information from the apical complex of PAR3, PAR6 and
atpical protein kinase C f (aPKC-f) and basolaterally localized
scribble, discs large (DLG) and lethal giant larva (LGL) (Feigin
and Muthuswamy, 2009).
Cancer in general, and breast cancer in particular, involves a
characteristic loss of apico-basal polarity, changes in cell–cell
adhesion, and a transition away from a simple highly polarized
epithelia to a multilayered incompletely polarized epithelia
(Rosen, 2001; Feigin and Muthuswamy, 2009; Huang and
Muthuswamy, 2010). Cell adhesion genes can act as tumor
suppressors in mouse models of mammary carcinoma and are
frequently mutated in human breast tumors (Berx et al., 1996;
Hirohashi, 1998; Conacci-Sorrell et al., 2002; Bogenrieder and
Herlyn, 2003; Knudsen and Wheelock, 2005; Derksen et al.,
2006; Jeanes et al., 2008; Derksen et al., 2011). Similarly, cell
polarity genes can act as tumor suppressors, their loss can
cooperate with oncogene function in model systems and their
expression and localization is frequently disrupted in human
cancers (Bilder et al., 2000; Pagliarini et al., 2003; Pagliarini
and Xu, 2003; Feigin and Muthuswamy, 2009; Huang and
Muthuswamy, 2010). However, it is unclear whether the changes
in cell–cell adhesion and loss of apico-basal polarity observed in
cancer are specific features of neoplastic growth or whether
normal epithelial morphogenesis also requires reduced polarity
In the present study, we sought to determine whether apico-basal
polarity and junctional adhesion were preserved in the multilayered
epithelium characteristic of mammary branching morphogenesis.
We analyzed the fine structure of mammary epithelium during
growth in a 3D organotypic culture model and then the in
vivo organization of TEBs during pubertal development. We
microscopy of cell and protrusion dynamics within the epithelial
multilayer during morphogenesis in 3D culture.
Mammary epithelial cells can form highly polarized
bilayers in Matrigel
We previously established culture conditions sufficient for
survival and differentiation of primary mammary epithelium
explanted into Matrigel (simple medium: DMEM with F12,
insulin-transferrin-selenium and penicillin-streptomycin) in which
essentially all epithelial fragments (‘organoids’) will form
bilayered cysts (Fata et al., 2007; Ewald et al., 2008). Addition
of nanomolar concentrations of growth factor induces a complex
multi-day program of branching morphogenesis (Fata et al., 2007;
Ewald et al., 2008). We selected FGF2 for this study as it induces
branching morphogenesis with high efficiency (80–95% of
organoids) in 3D culture and FGFR2 is required in the TEB
during puberty (Lu et al., 2008).
organization of bilayered mammary epithelium in Matrigel.
We examined 75 sections from five mammary cysts in
simple medium and observed simple epithelial organization by
ultrastructure (Fig. 1E). The lumens were electron dense and
contained secretory material (black arrows in Fig. 1E–E0). Cells
lining the lumen had microvilli (Fig. 1E9) and were connected by
tight junctions (Fig. 1E0). We also observed desmosomes
connecting luminal–luminal (Fig. 1E9), luminal–myoepithelial
(Fig. 1E0) and myoepithelial–myoepithelial cells (not shown).
The basal tissue surface was smooth and we detected no
protrusions into the ECM.
Collective epithelial cell migration 2639
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Morphological polarity can be assessed with transmission
electron microscopy (TEM), but molecular polarity is most
readily assayed with immunofluorescence. Simple epithelial
polarity is associated with apical localization of the PAR3–
PAR6–aPKC-f complex and basolateral localization of scribble.
Consistent with the high degree of morphological polarity
observed by TEM, the molecular polarity complexes were also
segregated into distinct apical and basolateral domains. PAR3
was apically localized on the lumen-facing surface of the luminal
epithelial cells (Fig. 1F), whereas scribble was basolaterally
localized in the luminal epithelial cells (Fig. 1G). The protein
numb is involved in cell polarity, adhesion (Wang et al., 2009)
and asymmetric cell division in multiple systems (Go ¨nczy, 2008).
In polarized bilayers its localization is confined to the basolateral
Fig. 1. Normal mammary morphogenesis is accomplished by a stratified epithelium. (A) Carmine-Red-stained 10-week-old mouse mammary gland.
Mammary ducts are elongated during puberty by specialized structures at the end of the duct, terminal end buds (TEBs). (B) Resting mammary ducts have a
bilayered organization, with luminal epithelial cells, connected by extensive intercellular junctions, and basally located myoepithelial cells. (C) Normal ducts in
vivo have a simple epithelial organization when not actively growing. Zona occludens 1 (ZO-1) is localized to the apico-lateral surface of the luminal epithelial
cells and b-catenin to the basolateral surfaces. (D) Mammary ducts are elongated during puberty by TEBs, stratified epithelial structures with many luminal cell
layers. b-catenin localizes to all basolateral surfaces and is only excluded from lumen-facing surfaces. ZO-1 localizes to the lumen lining surfaces of both the main
lumen and isolated micro-lumens. (E–E0 0) Primary mammary ducts can be isolated and grown in 3D Matrigel gels. Without the addition of growth factor all ducts
form simple cysts. These cysts are bilayered, with a single luminal (Lum) cell layer and a single myoepithelial (Myo) layer. The lumen has electron dense
secretory material (arrows), microvilli (MV), and tight junctions (TJ). Both luminal and myoepithelial cells are connected by desmosomes (Des). (F–H) Polarized
cysts in 3D culture localize PAR3 to apical surfaces (F) and both scribble (G) and numb (H) to basolateral surfaces. All TEM images are from high-pressure
frozen, freeze-substituted samples that were pre-fixed with 4% glutaraldehyde.
Journal of Cell Science 125 (11)2640
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domain (Fig. 1H). We therefore conclude that our epithelial
isolation and culture conditions in Matrigel are compatible with
complexes and fluid-filled lumens, consistent with the in vivo
organization of mammary ducts in the mouse (Pitelka et al.,
1973) and human mammary glands (Ozzello, 1971; Stirling and
Chandler, 1976; Stirling and Chandler, 1977).
Mammary morphogenesis in 3D culture is accomplished
via a transiently stratified epithelium
Mammary epithelium in 3D culture forms a multilayered
mammary buds (Ewald et al., 2008). Apico-basal polarity is
well defined in simple epithelia (Ozzello, 1971; Pitelka et al.,
1973; Nelson, 2009), but the extent of polarity within the interior
of a transiently multilayered epithelium was unclear. We have
previously shown that aPKC-f localizes to all interior contact
surfaces within the multilayer suggesting that interior cells lack
segregated apical and basolateral membrane domains (Fig. 2A)
(Ewald et al., 2008). Following morphogenesis, the localization
of aPKC-f in the re-established simple epithelium is restricted to
the apical surface (Fig. 2A9) (Ewald et al., 2008). We have also
shown that E-cadherin and b-catenin localize to all interior
contact surfaces within the multilayer, both in vivo and in vitro
(Fig. 2B–C9) (Ewald et al., 2008). Because E-cadherin and b-
catenin are major components of the adherens junction these data
Fig. 2. During morphogenesis in 3D culture the mammary epithelium is transiently stratified. (A–C9 9) Within the multilayered epithelium of elongating buds
in 3D culture, most interior cell surfaces are not in contact with the lumen or the ECM. These interior surfaces stained positive for aPKC-f (A), E-cadherin (B) and
b-catenin (C). Following morphogenesis, the mammary epithelium regained a simple organization. In regions of simple organization, aPKC-f localized to apical
domains (A9) and b-catenin localized to basolateral domains (C9). (D–E9 9) TEM was used to define the ultrastructural polarity of the tissue. The basal tissue surface
was smooth and lacks ECM-directed protrusions. (F–G- -) Both elongated cells (F’) and round unpolarized cells (F-) are present in the interior of the multilayer.
Away from the basal tissue surface, the epithelial cells exhibit dense, inter-digitating membrane extensions (F0). We also observed division of round cells distant
from either the ECM or lumen facing surfaces (F, green asterisk). (G–G- -) Within the same branching structure, there are regions with simple epithelial
organization with an electron-dense lumen and tight junctions. All TEM images are from high-pressure frozen, freeze-substituted samples that were pre-fixed with
4% glutaraldehyde. ED, electron dense; EL, electron lucid; TJ, tight junction.
Collective epithelial cell migration 2641
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motivated us to assay for junctional complexes directly. Here, we
used TEM and further molecular marker analysis to test our
hypothesis that mammary morphogenesis involves a transient
reduction in apico-basal polarity and junctional adhesion in
Mammary epithelium during morphogenesis lacks
protrusions into the ECM
A newly discovered feature of mammary collective epithelial
migration is the absence of forward-oriented actin protrusions in
advance of elongating ducts (Ewald et al., 2008). However, it was
possible that fine protrusions extended into the ECMthat could not
be detected by light microscopy. To test for fine protrusions, we
fixed organoids during active branching and identified regions of
active ductal elongation on the basis of the organization of the
epithelium. We examined 95 sections from 12 samples. We
typically observed ducts elongating simultaneously in multiple
directions (Fig. 2A–D). We then examined the basal surface of
elongating ducts in 3D culture at high resolution by TEM. We saw
no evidence of forward-oriented protrusions into the ECM (0 of 95
sections, Fig. 2D–F). Weinsteadobserved a smoothand organized
basal tissue surface. Individual cells showed polarized basal
surfaces at the tissue–ECM interface, but little morphologically
evident polarity on interior surfaces (Fig. 2F–F-).
Mammary epithelial morphogenesis in 3D culture involves
a reduction in intercellular junctions
Because cells in the interior of the multilayer did not have
conventional epithelial cell morphologies, we next sought to
determine the extent to which intercellular junctions are
observed well-defined apical tissue surfaces, with electron-
dense lumens, microvilli and extensive secretory material
(Fig. 2D,G–G-). Tight junctions only connected cells lining
electron-dense luminal spaces (TJ in Fig. 2G0,G-). We observed
an electron-dense luminalspace
intercellular space between cells in the multilayered region
(Fig. 2G0,G-). By contrast, cells within the interior of the
multilayered epithelium lacked most features of simple epithelial
organization or apico-basal polarity. We did not observe a zonula
adherens at points of interior cell–cell contact. However, E-
cadherin and b-catenin were still membrane localized in interior
cells and so we cannot rule out the possibility of small E-
cadherin-based adhesive complexes that would be difficult to
detect by TEM. Cell–cell contact in the interior appeared loose
and was characterized by extensive frequently interdigitating
membrane extensions (Fig. 2F0). Interior cells displayed a variety
of cell shapes and sizes and frequently included round cells with
minimal cell–cell contacts (Fig. 2F-).
Interior cells lack membrane-associated PAR3 and are
Because the interior cells lacked morphological polarity, we next
used antibodies to visualize the localization of molecular markers
for apico-basal polarity during each stage of morphogenesis in
3D culture. Interior cells within the multilayered epithelium
displayed a diffuse cytoplasmic localization of PAR3 (Fig. 3A).
By contrast, scribble was membrane-associated on all interior cell
surfaces in the multilayer and was absent only from lumen-facing
surfaces (Fig. 3B). Similar to the localization of scribble, numb
was localized to all lateral surfaces at the complex cyst stage. In
the multilayered epithelium of elongating ducts, PAR3 localized
to all lumen-facing membranes, but was diffusely localized to the
cytoplasm in interior cells (Fig. 3D). Scribble and numb instead
localized to all interior cell surfaces and were excluded from
lumen-facing surfaces (Fig. 3E,F). After morphogenesis, the
Fig. 3. Morphogenesis in 3D culture is associated with a loss of molecular polarity. (A–C) Interior cells are first evident in ‘complex cyst’ structures. Within
these structures interior cells display cytoplasmic localization of PAR3 (A), whereas scribble and numb localize to all interior cell surfaces (B,C). (D–F9 9) In the
multilayered region of elongating mammary end buds PAR3 is cytoplasmically localized (D), whereas scribble and numb localize to all interior surfaces.
After morphogenesis is complete the epithelium regains a simple epithelial organization and PAR3 is associated with the apical membrane (D9), whereas scribble
and numb are localized to basolateral cell surfaces (E9,F9).
Journal of Cell Science 125 (11)2642
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reformed simple epithelium had PAR3 that was localized to the
apical surface of luminal cells and scribble and numb to
basolateral surfaces (Fig. 3D9,E9,F9).
Isolated microlumens exist between interior cells in the
multilayered epithelium in 3D culture
We have previously observed small isolated zona occludens
protein 1 (ZO-1; also known as TJP1)-lined spaces within the
multilayered epithelium of elongating mammary buds both in 3D
culture and in vivo (Ewald et al., 2008). We sought to distinguish
whether these represent small lumens or accumulations of apical
molecular markers without corresponding ultrastructural features.
We first used confocal microscopy to collect optical sections and
generate 3D reconstructions of the multilayered mammary
epithelium during 3D culture. We discovered that the ZO-1-
lined microlumens were isolated from the main lumen of the
organoid (Fig. 4A–A0). Following morphogenesis, individual
ducts had single simple lumens (Fig. 4B,B9). Using TEM we
observed both empty (electron lucid) intercellular regions
(Fig. 4C9) and isolated lumens filled with electron-dense
material (Fig. 4C0–D-). The electron-lucid intercellular regions
had extensive membrane protrusions but no evidence of
tight junctions or secretory material (Fig. 4C9, white arrow).
Desmosomes were the only junctions that we observed
connecting cells surrounding the electron-lucid intercellular
spaces. By contrast, isolated microlumens had microvilli, as
well as tight junctions at points of apical intercellular contact and
accumulated electron-dense material (Fig. 4C0–D-). Isolated
lumens extended several microns into the multilayer (Fig. 4E).
Epithelial organization is highly variable during
morphogenesis in 3D culture
Our expectation was that cells at the basal tissue surface would
remain columnar throughout branching. Strikingly, this was not
the case and we observed considerable heterogeneity in the shape
of basally located cells (Fig. 5A), with ‘islands’ of high local
order (Fig. 5A9) and disorder (Fig. 5A0) located in the same
branching structure. Even in actively branching structures with
highly variable cell shapes, there were frequently locations with
a single luminal epithelial cell layer and highly polarized
ultrastructural organization. Following branching the transiently
stratified epithelium returned to a simple epithelial organization,
with mature apico-basal polarity at the morphological level.
Regions with few intercellular junctions display extensive
3D membrane protrusions
During active morphogenesis in 3D culture, we observed few
intercellular junctions connecting interior cells, but extensive
membrane protrusions. Some of these protrusions had the
(Fig. 5B,B9). However, membrane protrusions were observed at
some distance from the apical surface and were frequently
Fig. 4. The multilayered region contains
microlumens with tight junctions.
(A–A0 0) Light microscopy of the multilayered
region. ZO-1 (green) localizes both to the lining
of the main lumen and to isolated microlumens
located between luminal epithelial cells
(arrowheads in 3D volume reconstruction).
(B,B9 9) Following morphogenesis a simple
epithelial organization is restored and a single
clear lumen is observed. (C–D- -) Using TEM
there are two different intercellular spaces:
electron-lucid spaces with extensive irregular
membrane protrusions but without tight
junctions (C9, white arrows) and electron-dense
intercellular spaces with tight junctions (TJ),
microvilli and secretory material (red arrow in
C0) (C0–D-). (E) Serial block face scanning
electron microscopy enabled 3D reconstruction
of the microlumens and confirmed that they are
several microns thick. All TEM images are
from high-pressure frozen, freeze-substituted
samples that were pre-fixed with 4%
glutaraldehyde. The image series used to
generate E has been uploaded to ‘The Cell: An
Collective epithelial cell migration2643
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intermixed with desmosomes. Membrane protrusions were
several microns in length (Fig. 5C,C9) and branched multiple
times (Fig. 5D). The uniform thickness along the protrusions, as
well as the tendency for the protrusions to appear continuous over
long distances, led us to analyze whether they were thin
microvillar-like individual membrane protrusions or whether
they were complex 3D membrane extensions. Accordingly,
we collected a series of consecutive 90-nm sections and
imaged each by TEM (Fig. 5E,E9; supplementary material
Fig. S1A). Protrusions extended through 5–10 sections and
changed continuously from branched to unbranched (Fig. 5E9,
To confirm the 3D structure of these newly discovered
membrane protrusions we next collected continuous image series
using serial block face scanning electron microscopy (SFB-SEM)
and focused ion beam scanning electron microscopy (FIB-SEM).
In both cases, a thin layer of material was removed from the
surface of the embedded sample and the block face was then
imaged. The procedure was repeated to produce a 3D image series.
Serial block face and focused ion beam imaging both prevent
warping artifactsassociatedwith transferofathin section toa solid
support. Weobserved transitions from thin to thick protrusions and
from linear to branched protrusions (Fig. 5F; supplementary
material Fig. S1B, Fig. S2). We conclude that these membrane
elements are interdigitating 3D membrane extensions (Fig. 5G–
H0). By contrast, we did not observe complex 3D extensions in
regions of simple epithelial organization.
Interior cells exhibit apparently migratory polarity during
morphogenesis in 3D culture
As ductal elongation ceases, the multilayered duct reorganizes to
a simple epithelium (Ewald et al., 2008). In electron micrographs,
this corresponded with a transition from largely non-junctional
cell adhesion with loose cell–cell contact to a simple epithelial
Fig. 5. Cell shape and lateral membrane
organization are highly heterogeneous in the
multilayered region. (A–A0 0) Nearby regions in the
same branching structure can have high (A9) or low
(A0) epithelial organization. (B–D) Lateral
membrane protrusions within the multilayer were
frequently interdigitated (B–B9), could be several
microns in length (C–C9) and were frequently
branched (D). (E–F) Analysis of serial sections by
TEM (E,E9) and serial block face scanning electron
microscopy (F) revealed that the lateral membrane
protrusions extend through multiple sections (colored
arrowheads) and can morph between thin and broad
and between linear and branched. (G–H0 0) Three-
dimensional reconstructions of cell contact regions
along interior lateral surfaces using serial block face
SEM revealed densely interdigitating 3D membrane
extensions. All TEM images are from high-pressure
frozen, freeze-substituted samples that were pre-
fixed with 4% glutaraldehyde. The image series used
to generate F has been uploaded to ‘The Cell: An
Journal of Cell Science 125 (11)2644
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organization with extensive intercellular junctions. One possible
mechanism for a transition from a multilayered to simple
organization is radial intercalation, in which cells move
between layers to give a thinner tissue (Keller, 2002; Stubbs
et al., 2006). We observed elongated cells within the multilayered
epithelium whose morphology was consistent with individualistic
cell migration (Fig. 6A–D). Despite the absence of actin-based
protrusions extending into the ECM, we observed long extensions
at the front of these elongated cells (Fig. 6A,B). One cell that
appeared to have recently arrived at the basal surface displayed
an intermediate polarity between elongated and apico-basal
Desmosomes are frequently observed in the interior of the
We have demonstrated that a subset of cells in the interior of
the multilayered epithelium lack molecular apico-basal polarity
and appear to have a migratory polarity and forward-oriented
Fig. 6. Elongated cells are observed within the epithelial multilayer. (A–A0 0) Electron-dense elongated cells are seen within the multilayered region. These
cells frequently have long protrusions extending in a single direction between adjacent cells (A0). (B–C9 9) Elongated cells were observed deep within the
multilayer (A) and also within microns of the ECM (B). The only intercellular junctions observed on these cells were small desmosomes (C–C9). (D–D0 0) At the
ECM border some cells had an appearance that was intermediate between columnar epithelial and elongated morphologies. They did not extend protrusions into
the ECM. (E–F9 9) Cells in extensive contact with the ECM had smooth basal surfaces and lateral desmosomes, but frequently displayed little morphologic polarity
on their lateral surfaces. (G–H) Interior cells have lateral surfaces with intermixed membrane protrusions and small desmosomes. All TEM images are from high-
pressure frozen, freeze-substituted samples that were pre-fixed with 4% glutaraldehyde.
Collective epithelial cell migration2645
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dissemination of luminal epithelial cells in 3D culture in the
hundreds of movies wecollected inour previous study (Ewald etal.,
2008). We therefore sought to identify persistent intercellular
junctions that might limit the dissemination of luminal epithelial
cells. The only intercellular junctions that we regularly observed
connecting interior cells during morphogenesis were desmosomes
(Fig. 6B9–C9,E–H).We observed
apparently migratory cells to their neighbors in close proximity to
the ECM (Fig. 6B–C9). We also observed desmosomes connecting
columnar cells at the ECM border (Fig. 6E–F9) and interior cells
within the multilayer (Fig. 6G,H). Desmosomes in the interior were
small and did not accumulate extensive intracellular plaques to the
degree observed in highly polarized epithelial cells (compare
Fig. 6G,H with Fig. 1E9,E0).
However,wedid not observe spontaneous
Interior cells are protrusive and migratory within the
Our electron micrographs revealed extensive protrusions and
apparent migratory polarity in cells within the epithelial
multilayer. However, it is difficult to infer cell movements
accurately from a series of fixed specimens. We therefore labeled
small numbers of cells using adenovirally delivered cytoplasmic
GFP and imaged their behavior using time-lapse confocal
microscopy. We tracked 21 cells from 10 movies collected
over three independent experiments. All 21 cells were protrusive
and motile. Cells had a mean speed of 5.561.8 mm per hour
We observed individual cells migrating over multiple cell
diameters withinthe multilayered
supplementary material Movie 1). Round cells in the interior
extended and retracted long cytoplasmic protrusions (Fig. 7B;
supplementary material Movie 2). We also directly observed
radial intercalation of front–rear-polarized migratory cells into
the tissue basal surface (Fig. 7C; supplementary material Movie
3). The forward extensions in these cells immediately flattened
out as the cells made contact with the extracellular matrix. Cells
just inside from the tissue basal surface extended and retracted
protrusions over most of their cellular surface area (Fig. 7D;
supplementary material Movie 4). Cells bridging tissue apical
and tissue basal surfaces also extended and retracted protrusions
along their lateral surfaces (Fig. 7E; supplementary material
Movie 5). We did not observe protrusions into the ECM or the
lumen. We conclude that mammary epithelial cells display
extensive protrusions during morphogenesis in 3D culture on all
interior cell surfaces and that individual cells appear to migrate
actively in the interior of the multilayer.
Fig. 7. Interior cells are frequently
migratory and protrusive within the
multilayer. All cells were labeled
with Cell Tracker Red and a subset of
cells were labeled with an
adenovirally delivered cytoplasmic
GFP. Images depict 3D
reconstructions of the cytoplasm in
individual GFP-positive cells.
(A) Interior cells were frequently
highly migratory and could move in
directions opposite to the direction of
ductal elongation. (B) Interior cells
extended and retracted cytoplasmic
protrusions within the multilayer, but
not into the lumen or ECM.
(C) Individual cells migrated from the
interior to basal positions in contact
with the ECM, a process termed radial
intercalation. (D,E) Cells in contact
with the basal tissue surface (D) or
both apical and basal tissue surfaces
(D,E) extended cytoplasmic
protrusions from all lateral surfaces.
The movies used to generate A–E are
presented in supplementary material
Movies 1–5, respectively.
Journal of Cell Science 125 (11) 2646
Journal of Cell Science
Inhibition of MLCK or Rac blocks duct initiation but results
in different architectural states
Pharmacological inhibition of either Rac or myosin light chain
kinase (MLCK) results in a dose-dependent block of ductal
initiation (Ewald et al., 2008). In both cases, when we added the
inhibitor at the start of culture the lumens fill as normal, but new
ducts do not initiate. However, inhibition of Rac results in a
persistently multilayered epithelium, whereas MLCK-inhibited
structures eventually clear their lumens to form a simple
epithelial organization (Ewald et al., 2008). Accordingly, we
examined organoids treated with 100 mM Rac inhibitor and
collected electron micrographs to determine whether these
structures were well polarized (two samples, 220 sections
imaged;supplementary materialFig. S3).We observed
extensive cell–cell contact through interdigitating membrane
protrusions. By contrast, organoids treated with 1 mM MLCK
ultrastructural organization was indistinguishable from simple
cysts (one sample, 16 sections imaged; supplementary material
lumensand their final
ROCK inhibition prevents reversion to the single layered
state and results reduced interior cell-cell contact
Pharmacologic inhibition of Rho kinase (ROCK) with Y-27632
results in a hyper-branched phenotype with reduced levels of E-
cadherin at intercellular surfaces (Ewald et al., 2008). On the basis
of the lower intensity and punctate appearance of E-cadherin
staining following inhibition of ROCK, we hypothesized that
Fig. 8. Treatment with Y-27632 results
in disorganization and reduced cell–
cell contact on lateral and apical
surfaces. (A–C9 9) Normal organoids have
a stereotyped branching pattern,
with a large ZO-1-lined lumen
(A,A9). Treatment with the ROCK
inhibitor Y-27632 results in rapid loss of
the lumen, luminal epithelial
disorganization and localization of ZO-1
exclusively to small foci (B,B9). Large
regions of Y-27632-treated epithelium
were free of ZO-1 immunoreactivity (red,
phalloidin; green, ZO-1; blue, nuclei).
(D–H- -) Ultrastructural examination
revealed a loss of electron-dense luminal
spaces and a large decrease in cell–cell
contact along lateral cell surfaces. The
basal tissue surface was still smooth and
well organized (H–H-). The only
junctions we observed connecting cells in
Y-27632-treated samples were small
desmosomes (red asterisks, E9,E0,G9,G0).
All TEM images are from high-pressure
frozen, freeze-substituted samples that
were pre-fixed with 4% glutaraldehyde.
(I,I9 9) Organoids treated with the ROCK
inhibitor H1152 had microlumen-
localized PAR3 (I, 3D reconstruction; I9,
2D optical section). (J,J9 9) Organoids
treated with H1152 had lateral-surface-
localized scribble but no scribble on
the microlumen surface (J, 3D
reconstruction; J9, 2D optical section).
Collective epithelial cell migration2647
Journal of Cell Science
Y-27632 treatment disrupts cell–cell contacts between luminal
epithelial cells. Indeed, we observed dramatic changes in the
localization of ZO-1 in ROCK-inhibitor-treated organoids
(compare Fig. 8B–C9 with Fig. 8A,A9). Following ROCK
inhibition, ZO-1 was frequently associated with small puncta
(Fig. 8B9), but large regions that lacked ZO-1 immunoreactivity
werealso observed(Fig. 8C9).
observations, we observed a striking reduction in intercellular
contact in TEM images of ROCK-inhibited organoids (Fig. 8D–
H-; two samples, 34 sections imaged). Lumens were no longer
observed and cell–cell contacts were barely maintained on lateral
surfaces (Fig. 8D,F). We essentially observed a complete loss of
luminal organization with no tight or adherens junctions. Instead
of electron-dense lumens, there were electron-lucid intercellular
spaces. Interior cells barely contacted each other and appeared
completely unpolarized. Despite this dramatic loss of polarity and
the reduction in cell–cell contact, we still observed occasional
small desmosomes (Fig. 8E9,E0,G0) and a well-organized basal
tissue surface (Fig. 8H–H-). This dramatic reduction in polarity
and adhesion is reversible (Ewald et al., 2008) and was not
Consistent with these
sufficient for dissemination of interior cells into the ECM.
Treatment with H1152, an alternate ROCK inhibitor, also
induced loss of the main lumen and extensive microlumens
(Fig. 8I,J; supplementary material Fig. S4). However, some
apico-basal polarity was maintained in H1152-treated organoids
as PAR3 localized to the membrane of the microlumens
(Fig. 8I,I9), whereas scribble localized to basolateral surfaces
and was excluded from the microlumens (Fig. 8J,J9). We
conclude that ROCK (or another Y27632-sensitive kinase) is
required for maintenance of luminal continuity and lateral cell–
cell adhesion within the luminal epithelial cell layer.
Terminal end buds in vivo have similar organization to
mammary epithelium during branching morphogenesis in 3D
culture, we next asked whether the morphology of the interior
cells of branching organoids matched the organization of interior
(‘body’) cells of the normal in vivo structure for mammary ductal
elongation, the TEB. We collected electron micrographs of
ultrastructural organization of
Fig. 9. TEBs in vivo display
reduced apico-basal polarity and
extensive intercellular membrane
protrusions. (A) Mammary epithelial
ducts are elongated by TEBs, shown
here in a light micrograph, with boxes
indicating regions that were
subsequently imaged by TEM.
(B,B9 9) TEBs contain a fluid-filled
lumen with microvilli and tight
junctions. (C–F0 0) Cells within the
multilayered region appeared
morphologically unpolarized and
displayed extensive intercellular
protrusions. These protrusions were
observed to interdigitate (D) and
branch (F0). (G–G0 0) Cells at the
apical or basal most tissue surface
were polarized at the tissue boundary
but could be unpolarized and
irregularly shaped a few microns
away. (H) Microlumens within the
body cell region had both microvilli
and tight junctions. All TEM images
are from high pressure frozen, freeze-
substituted samples that were pre-
fixed with 4% glutaraldehyde. Des,
desmosomes; TJ, tight junctions.
Journal of Cell Science 125 (11) 2648
Journal of Cell Science
sections through TEBs, including regions at the cell–ECM,
lumen-facing and interior surfaces (Fig. 9; four samples, 60
sections imaged). Consistent with our in vitro results, we
observed tight junctions connecting cells at surfaces facing
lumens (Fig. 9B,B9). We also did not observe protrusions from
the basal TEB surface into the ECM. As in the 3D cultures, there
was considerable heterogeneity in cell and nuclear morphologies
in the TEB (Fig. 9C). Body cells of TEBs had a similar cellular
organization to interior cells in 3D culture, with extensive
interdigitating lateral membrane protrusions (Fig. 9D–F0). The
TEB was polarized at its apical and basal most tissue surfaces,
with many morphologically unpolarized cells in the interior
(Fig. 9C–F). Furthermore, the morphological polarity of cells
lining the lumen was generally restricted to the lumen-facing
surface, with interior surfaces adopting a round or mesenchymal
morphology (Fig. 9G–G0). We also observed microlumens within
the multilayered region of the TEB, both by light (Fig. 1D) and
electron microscopy (Fig. 9H).
The mammary gland undergoes multiple rounds of epithelial
involution (Hennighausen and Robinson, 2005; Sternlicht,
2006). The mammary epithelium
organization, with a single luminal epithelial cell layer
(Ozzello, 1971; Pitelka et al., 1973). During early embryonic
development and during puberty the elongating tips of mammary
ducts are multilayered (Hogg et al., 1983; Williams and Daniel,
1983; Ewald et al., 2008). In this study, we used molecular
markers and TEM to determine the polarity and adhesional status
of this multilayered intermediate. Interior cell surfaces within the
multilayered localized both apical (aPKC-f) and basolateral
(scribble) molecular markers. Despite E-cadherin and b-catenin
localization, we did not observe zonula adherens connecting
interior cells. Instead points of cell–cell contact in the interior
were connected by desmosomes and characterized by extensive
interdigitating 3D membrane extensions. Interior cells were
observed to extend protrusions and to migrate, but these
behaviors were transient and spatially limited to the interior of
the multilayer (Fig. 10).
at rest has asimple
3D primary organotypic culture recapitulates the normal
junctional adhesion of the mammary epithelium
Without growth factor stimulation primary mammary organoids
in 3D culture robustly form cysts with a highly polarized
bilayered architecture and microvilli lining the lumen. The
epithelium developed a complete apical junctional complex and
displayed extensive desmosomal connections along lateral
surfaces. This organization is consistent with the normal
ultrastructure of the mammary epithelium in vivo (Pitelka et al.,
1973), and is in contrast to the ultrastructural organization of
MCF-10A acini cultured under similar 3D culture conditions.
MCF-10A cysts do not form detectable tight junctions and
instead appearto formtheir
desmosomal contacts (Underwood et al., 2006). Our primary
3D cultures therefore provide a starting point for mechanistic
evaluation of the relative importance of different junctional
complexes and adhesion proteins.
lumens through extensive
Mammary epithelium adopts a low polarity state with few
junctions during morphogenesis
During morphogenesis, the mammary epithelium was polarized at
its apical (lumen-facing) and basal (ECM-contacting) tissue
surfaces. However, even cells at the lumen or ECM border
typically were polarized only on their apical- or basal-most cell
dense interdigitating membrane protrusions. Some of these
protrusions appeared morphologically similar to microvilli,
intermingled with desmosomes in electron-lucid intercellular
spaces, were several microns long, or were branched, all features
uncharacteristic of classical microvilli. In volume reconstructions,
these protrusions appeared as an interconnected 3D network of
membrane extensions, rather than individual thin projections.
Because we observed similar membrane protrusions on interior
cells in TEBs from normal mammary glands, we conclude that
these structures are transiently produced both in vivo and in vitro
during normal mammary morphogenesis. These 3D membrane
extensions increase theentangled surface area of adjacent cellsand
we speculate that they might thereby assist in maintaining
intercellular adhesion among motile cells.
Other protrusions were
Tight junctions are only observed lining lumens
Tight junctions classically serve two functions: gate and fence.
They function as a gate to enable selective control over
paracellular permeability. They also function as a fence within
the membrane, partitioning the apical from basolateral membrane
regions (Nelson, 2009). Previous work has established that TJs
can exist in stratified epithelia (Langbein et al., 2002), but we
found no evidence for TJs in the interior of the multilayer during
morphogenesis except at the apical end of cells facing lumens.
Without TJs in the interior of the multilayer, it is consistent that
the membranes appear to have an unresolved mix of apical and
Mammary epithelial cells display extensive protrusive
activity and individualistic cell migration within the
Elongating mammary buds maintain a smooth and highly
organized basal-tissue–ECM surface both in vivo and in 3D
culture. However, we observed extensive protrusions in the
interior of the epithelium. We did not observe protrusions into the
ECM at either the light or electron microscopy levels. These
observations imply that there is a reduction in the epithelial
Fig. 10. Normal mammary epithelial morphogenesis is
accomplished via a transiently stratified epithelium. The
transient multilayered epithelium associated with mammary
morphogenesis is polarized as a tissue but displays reduced
apico-basal polarity and few intercellular junctions at points
of cell–cell contact in the interior.
Collective epithelial cell migration2649
Journal of Cell Science
character of mammary epithelial cells during morphogenesis, as
protrusive migration is characteristic of mesenchymal cells.
Strikingly, this reduction was spatially restricted to the
multilayered region within elongated ducts and was reversible,
with normal ducts eventually reverting to bilayered organization.
Epithelial organization during morphogenesis is similar in
3D culture and in vivo
The low intercellular adhesion, reduced polarity environment that
we observed during morphogenesis in organotypic culture is
highly similar to the organization of the in vivo mammary
terminal end bud (TEB). The essential features of reduced
intercellular adhesion, reduced apico-basal polarity, fewer
intercellular junctions and stratified epithelial organization are
all quite similar between organoids and TEBs. We observed tight
junctions at the apical end of cells facing microlumens, and
desmosomes connecting cells in the electron lucid intercellular
spaces in both TEBs and ducts in 3D culture. Significantly, the
low polarity environment is also similar to that observed in the
mammary placode during embryonic development (Hogg et al.,
1983; Nanba et al., 2001). Whereas our data directly address only
mouse mammary morphogenesis, immunohistochemistry of
sections from pubertal human mammary epithelium, and
human TEBs, indicates that a similar multilayered intermediate
underlies human mammary morphogenesis (Rudland, 1991).
There are extensive previous TEM studies of simple bilayered
mammary ducts (Ozzello, 1971; Pitelka et al., 1973), but we are
during puberty (Williams and Daniel, 1983). In that study the
authors observed membrane protrusions into intercellular spaces
between interior cells within the TEB, consistent with our results.
More recent TEM studies in the mammary gland have proposed
that small light-staining cells are stem or progenitor cells (Smith
and Medina, 1988; Chepko and Smith, 1997; Chepko and Smith,
1999; Smith and Chepko, 2001). We also observed variation in
electron density, with smaller rounder cells typically staining more
lightly, and larger elongated cells typically staining darker.
Collective epithelial migration has features of the epithelial
to mesenchymal transition
A primary model for the cellular mechanism of normal and
neoplastic epithelial morphogenesis
mesenchymal transition (EMT). Morphological hallmarks of
this model include a reduction in epithelial organization and
acquisition of motility. EMT has been proposed to explain the
dissemination of previously adherent epithelial cells into the
surrounding ECM and is thought to be a major cellular
mechanism of metastasis in human breast cancer (Kalluri and
Weinberg, 2009; Polyak and Weinberg, 2009). We did not
observe migration of interior cells into the ECM either in 3D
culture or in vivo. However, the reduced epithelial polarity and
intercellular adhesion and increased motility that we observed
during morphogenesis are consistent with models of a partial
transient EMT (O’Brien et al., 2002). However, we observed a
striking confinement of these migratory cells to the interior of the
Furthermore, most models propose that EMT occurs at the
epithelial–stromal or tumor–stromal border (Lee et al., 2011). We
see the opposite; the highest level of E-cadherin staining and the
ultrastructurally highest degree of epithelial organization is at the
luminal and basal surfaces of the tissue. It is the interior
multilayered epithelial compartment, which is out of contact
with ECM and stromal cells, that exhibited the largest reduction
in epithelial character. Furthermore, the cellular phenotype
associated with inhibition of ROCK suggests that the luminal
and lateral intercellular adhesions can be essentially eliminated
without dissemination or loss of basal tissue organization.
Desmosomes are the most common junction connecting
cells in the interior of the multilayered epithelium
Research in epithelial biology has focused on the adherens
junction in part because E-cadherin is required very early in
embryonic development (Larue et al., 1994) and disruption of the
adherens junction can lead to disruption of the other adhesive
junctions (Gumbiner et al., 1988). However, disruptions in
desmosomal adhesion can also have severe phenotypes and can
alter the function of classical cadherin junctions (Green and
Gaudry, 2000; Vasioukhin et al., 2001; Den et al., 2006; Lechler
and Fuchs, 2007). Desmosomes were the main intercellular
junction that we observed in the interior of the multilayered
epithelium both in 3D culture and in vivo. Desmosomes were
also the only junction we observed connecting elongated,
apparently migratory cells to their neighbors. A previous TEM
study of cells in the interior of the elongating embryonic
mammary bud found very few junctional complexes connecting
interior cells, but did observe desmosomes (Hogg et al., 1983).
The requirement for desmosomal adhesion has not been tested
genetically in the mammary gland, but 3D culture studies suggest
that desmosomal cadherins are essential to establish and maintain
the correct apico-basal positions of luminal and myoepithelial
cells (Runswick et al., 2001). Furthermore, desmosomal
components are frequently mutated or silenced in breast cancer
(Klus et al., 2001; Oshiro et al., 2005), and both p53 and p63 can
regulate desmosomal adhesion (Ihrie and Attardi, 2005; Cui et al.,
2011; Dusek and Attardi, 2011).
Epithelial polarity and adhesion are altered during
Both normal and neoplastic mammary epithelia lose polarity,
reduce adhesion and shift from a simple to a multilayered
organization. Importantly, normal mammary epithelia reduce the
amount of polarity and adhesion in a highly restricted space for a
limited period of time, whereas mammary carcinomas persist in a
state with lower polarity and adhesion. The reversibility of even
very large changes in adhesion and polarity in normal epithelia
suggest that tumors might also be able to ‘correct’ their
organization and revert to a normal tubular architecture given
the right set of molecular signals. In fact, previous work spanning
over several decades has shown that human tumors can be
‘normalized’ by contact with embryonic mesenchyme and by
contact with cell-type specific extracellular matrix proteins
(Streuli et al., 1991; Petersen et al., 1992; Weaver et al., 1997;
Nelson and Bissell, 2006; Hendrix et al., 2007; Ingber, 2008;
Weigelt and Bissell, 2008).
A previous ultrastructural
hyperplasias, ductal carcinoma in situ and infiltrating ductal
carcinomas from human patients to determine the basis of the
invasive switch in human breast cancer (Goldenberg et al., 1969).
Importantly, the authors reported that: ‘‘The most notable finding
in ourultrastructural studyof varioustypes ofbreastlesionswasan
increased degree of cell membrane anomaly paralleling the
increased degree of aggressiveness among these lesions…
Journal of Cell Science 125 (11)2650
Journal of Cell Science
atypical ductal hyperplasia was characterized by abundant
microvilli… [i]ntraductal carcinomas were recognizable by
extremely complex cell interdigitations. Infiltrating ductal
carcinomas were detectable by an overabundance of cellular
microvilli’’ (Goldenberg et al., 1969). The morphological
similarity between the complex interdigitations in the intraductal
carcinomas in that study and in the normal mammary cells
undergoing morphogenesis in our study is striking and suggests
that at least some human breast cancers might recapitulate aspects
of a normal developmental migration program. We have
demonstrated in the present study that the ultrastructural
morphogenesis is highly similar to that during embryonic
morphogenesis (Hogg et al., 1983). This observation provides an
ultrastructural framework for the hypothesis that there is an
embryonic program of partial EMT that can be reactivated during
postnatal development and during cancer progression (Yang and
Normal mammary morphogenesis involves a shift from a simple
to a multilayered organization, dynamic cell movements and
reduced apico-basal polarity. In this study, we have defined a
new ultrastructural basis for the ‘morphogenetically active
epithelial state’ adopted during developmental remodeling of
the mammary gland. It is now important to understand the nature
of the molecular signals that enable this transition to be highly
regulated in space and time during normal development. In
particular, cell–cell adhesion
morphogenesis, with few observed intercellular junctions and
extensive entangled membranes. At present we do not know the
relative contributions of classical and desmosomal cadherin
systems to cell–cell adhesion during mammary morphogenesis.
Another major challenge is to define the cellular and molecular
mechanisms by which polarized epithelial cells reversibly create
a multilayered epithelium with lower levels of polarity.
is strikingly altered during
Materials and Methods
We generated epithelial fragments (‘organoids’) as previously described (Ewald
et al., 2008; Ewald, 2010), using 5- to 8-week-old Charles River FVB mice as the
source material. For ease of dissection, TEBs were isolated from a 5-week-old
fluorescent reporter transgenic mouse (Sca-1::EGFP) (Hanson et al., 2003).
Animals were housed and handled in accordance with approved IACUC protocols
at UCSF or JHU. Briefly, to generate organoids we surgically isolated the
mammary glands, minced them ,50 times with a scalpel and dispersed the glands
for 30 minutes in 50 ml collagenase solution at 37˚C, shaking at 100 rpm. The
resulting mixture was then centrifuged at 520 g for 10 minutes at 25˚C. We
aspirated away the supernatant and resuspended the pellet thoroughly in 4 ml
DNase solution. We shook the tube by hand for 2 to 5 minutes at room temperature
and then added 6 ml DMEM F12. We then centrifuged at 520 g for 10 minutes at
25˚C and aspirated the supernatant. To separate the epithelial organoids from the
single cells we used differential centrifugation, a total of 4 times. For each round
we resuspended the pellet thoroughly in 10 ml DMEM F12 and then pulsed the
tube to 520 g (typically 33 seconds). The resulting pellet was then resuspended in
the desired volume of growth factor reduced Matrigel (BD Biosciences) and plated
in 24-well not tissue-culture treated plates (Falcon). Organoids were grown for 5–6
days and then fixed during the time active branching morphogenesis was ongoing.
Solutions and reagents for organotypic culture
DMEM (Dulbecco’s modified Eagle’s medium complete) F12 (Gibco). Collagenase
solution consisted of: DMEM F12, fetal bovine serum (FBS, heat inactivated) (5%
final), gentamicin (50 mg/ml), insulin (5 mg/ml final, Sigma), trypsin (2 mg/ml,
Gibco), collagenase A (2 mg/ml, Type IV from Clostridium histolyticum, Sigma).
DNase I (Sigma) was resuspended at 4 U/ml in DMEM F12. Bovine serum albumin
(BSA; Invitrogen) was resuspended at 2.5 mg/ml in D-PBS. Simple medium:
DMEM F12, 16Pen-Strep, 16ITS (insulin, transferrin, sodium selenite; Sigma).
Branching medium: simple medium plus 2.5 nM FGF2 (Sigma). ROCK inhibitor
(Y27632; Chemicon) and Rac1 inhibitor (NSC23766; Calbiochem) were used as
indicated. All inhibitor experiments were done in FGF2 medium.
Fixation strategy for ultrastructural analysis
We initially utilized conventional chemical processing and transmission electron
microscopy (TEM) and observed minimally adherent cells, with highly
convoluted membranes and extensive intercellular spaces. However, we
encountered inadequate and irregular cytoplasmic and membrane preservation
(not shown). These limitations in tissue preservation are often associated with
room temperature dehydration and can be overcome by high-pressure freezing
and subsequent freeze substitution (McDonald and Auer, 2006). However, fresh
tissue microdissection and the exposure of unfixed tissues to relatively high
levels of cryoprotectants (e.g. 20% glycerol), just prior to high-pressure freezing,
can lead to physical damage or osmotic stress (Triffo et al., 2008). To avoid
aggregation artifacts caused by the dehydration step in conventional protocols,
yet guard against osmotic stress in fresh dissected tissue sample upon exposure to
cryoprotectants, we adopted a conservative approach to sample preparation. We
first prefixed in glutaraldehyde, then microdissected prefixed samples, then
processed the samples for high-pressure freezing and freeze substitution
(Sosinsky et al., 2008). Using this combination we were able to achieve high-
quality ultrastructural preservation across large sample regions. On separate
samples, we also employed serial block face SEM to allow 3D reconstruction of
selected regions of interest to identify the intercellular membrane protrusions.
Our focus was on the membrane organization and junctional complexes, both of
which were well preserved in our samples. Given the highly heterogeneous
epithelial environment in our samples, it was crucial to be able to process and
image large areas of the samples to enable both low-magnification surveys of
fields of cells and higher-resolution imaging of individual points of cell–cell
contact. Three biologically independent sets of samples were examined for the
main FGF2-treated branching organoids. Parallel samples were examined with
conventional chemical processing TEM. The conclusions were consistent across
all samples, but membrane preservation was better with high-pressure freezing
and freeze-substitution prepared samples. All images presented are from samples
subjected to high-pressure freezing.
Samples were pre-fixed overnight in 4% glutaraldehyde in organoid medium. They
were then subjected to high-pressure freezing in a BAL-TEC HPM-010 high-
pressure freezer (2100 bars for 5–7 milliseconds) (BAL-TEC, Carlsbad, CA) using
10% glycerol (v/v in cell culture medium) as the cryoprotectant in 200-mm deep
Using the Leica automated freeze-substitution system AFS (Leica Microsystems,
Vienna, Austria), cryofixed specimens were freeze-substituted in anhydrous
acetone containing 1% osmium tetroxide and 0.1% uranyl acetate and after several
rinses in pure acetone infiltrated with Epon-Araldite following established
protocols (McDonald and Mu ¨ller-Reichert, 2002; McDonald, 2007; Triffo et al.,
2008). Specimens were flat-embedded between two microscopy slides and
polymerized at 60˚C over 1 to 2 days. Resin-embedded samples were remounted
under a dissecting microscope for precise orientation.
Transmission electron microscopy
70–100-nm sections were collected on formvar-coated grids using a Reichert
Ultracut E ultramicrotome (Leica). Sections were post-stained using 2% uranyl
acetate in 70% methanol followed by Sato’s lead citrate. The sections were imaged
in an FEI Tecnai 12 TEM (FEI, Eindhoven, The Netherlands) operated at 120 kV.
Montaged TEM Images
Owing to the large size of the samples, it was frequently necessary to collect
multiple TEM images of overlapping adjacent areas to cover the relevant field of
view at the necessary resolution. Montages of electron microscopy images were
reconstructed using the freely available TrakEM2 program (Cardona et al., 2010;
Saalfeld et al., 2010; Schmid et al., 2010). Images were aligned at full 2048 by
2048 pixel resolution and contrast was adjusted across adjacent images using
TrakEm2. A custom Python script was written to assist in reconstruction. The
montage function in TrakEM2 was applied, with further manual alignment as
necessary. Overlaps were linear blended, and montages were exported as tiff files.
Serial block face and focused ion bean SEM sample preparation
Mouse mammary organoids were chemically fixed with 2.5% glutaraldehyde. To
produce enough back-scatter electrons for SBF-SEM imaging, the organoids were
stained with an osmium-thiocarbohydrazide-osmium (OTO) method (Friedman
and Ellisman, 1981; Willingham and Rutherford, 1984), in combination with
microwave-assisted processing. Organoids were rinsed three times with 0.1 M
sodium cacodylate buffer and then incubated with solution of reduced 2% osmium
Collective epithelial cell migration2651
Journal of Cell Science
tetroxide (containing 1.5% potassium ferricyanide) in buffer. The samples were
incubated using a Pelco Biowave microwave (Ted Pella Inc., Redding, CA) for 2
minutes at 150 watts of power. Following three rinses with buffer, the organoids
were microwave for 40 seconds at 150 W in 0.1% thiocarbohydrazide in double-
distilled water and then rinsed three times with water. Finally, they were
microwaved for 1 minute at 150 watts with 2% osmium tetroxide and rinsed three
times in water. To enhance preservation and contrast, the samples were high-
pressure frozen as described for the TEM samples and freeze substituted with a
solution of 4% osmium tetroxide, 0.1% uranyl acetate and 5% water in acetone.
Following five rinses in pure acetone samples were infiltrated with hard-forming
Epon resin with accelerator according to the following schedule: 2 hours in 2:1
acetone:resin; 2 hours in 1:1; 4 hours in 1:2; and overnight in pure resin. Samples
were flat-embedded as described for the TEM samples. Representative SBF-SEM
and FIB-SEM data series have been uploaded to the ‘The Cell: An Image Library’
(run by the ASCB; http://www.cellimagelibrary.org/).
Serial block face imaging
Organoids embedded in resin were mounted onto an aluminum pin with a
cyanoacrylate adhesive. The pin, which takes the place of a normal SEM stub, was
loaded into a sample holder for the Gatan 3View (Gatan, Pleasanton, CA). Serial
block face scanning electron microscopy was carried out as previously described
(Denk and Horstmann, 2004). Data were collected using an FEI Quanta 600 FEG
SEM; serial images were 4k by 4k and acquired at 5 kV; z-dimension slices of 50
nm. 3D volume representations of the data were prepared using CHIMERA
(Pettersen et al., 2004). CHIMERA was used to perform manual segmentation of
membranes and to display the 3D volumes.
Focused ion beam SEM imaging
Resin-embedded samples were trimmed with a thin razor blade to expose the area
of interest on both the top and one side of the block. This was then glued to a SEM
stub using colloidal silver paint. Milling and imaging of the block was carried out
using a FEI Strata 235 Dual Beam FIB (FEI, Hillsboro, OR). FIB milling at 50 pA
generated a beam size of ,17 nm. 1k by 1k images were collected with a
backscatter electron detector at 5 kV.
Staining for E-cadherin (Zymed, 13-1900), ZO-1 (Chemicon, MAB1520), SMA
(Sigma, F3777 or C6198), b-catenin (Santa Cruz Biotechnology, sc-7199), and
aPKC-f (Santa Cruz Biotechnology, sc-216) was performed as previously reported
(Ewald et al., 2008). Briefly, organoids were equilibrated in 25% sucrose in PBS
for 1 hour, fixed in cold 1:1 methanol:acetone overnight at –20˚C, then re-
equilibrated in 25% sucrose in PBS for 1 hour. Samples were blocked 1 hour with
5% serum, incubated with primary antibody (all 1:250 in PBS) for two hours to
overnight and rinsed three times in PBS. For PAR3 (Millipore, catalog number
07-330), numb (Cell Signaling Technology, C29611) and scribble (Santa Cruz
Biotechnology, sc-11049) staining, organoids were fixed in 4% PFA for 20
minutes and permeabilized in 0.5% Triton X-100 for 30 minutes. Samples were
blocked in 10% serum for 3 hours, incubated with primary antibody (all 1:500 in
10% serum) for two hours to overnight and rinsed three times in 10% serum.
Secondary Alexa-Fluor-conjugated antibodies (Molecular Probes, all 1:250 in
PBS) were incubated with the organoids for 1–4 hours. Nuclei were stained with
DAPI or propidium iodide (1:1000) (Molecular Probes). Antibody stains were
performed at least three independent times, inspecting a minimum of 25 organoids
in each condition each time.
Confocal imaging was performed on a Solamere Technology Group spinning disk
confocal microscope as described previously (Ewald, 2010), with a 406 C-
Apochromat objective lens (Zeiss Microimaging). Acquisition of both fixed and
time-lapse images was performed using a combination of mManager (Edelstein
et al., 2010) and Piper (Stanford Photonics). Levels were adjusted in Adobe
Photoshop to maximize clarity of the images. Level adjustments were always done
on the entire image.
Single-cell labeling with adenoviral GFP
Epithelial fragments were prepared using the previously described organotypic
culture method. Before resuspension in Matrigel 1000 organoids in 500 ml DMEM
F12 were transferred to a 1.7 ml Eppendorf tube. The tube was then pulsed at 520 g
for 30 seconds. Medium was removed from the pellet and organoids were
resuspended in 50 ml DMEM F12. Ad-CMV-GFP (Vector Biolabs, 1060) was then
added at a ratio of 2000 or 10,000 plaque-forming units (pfu):organoid. Epithelial
fragments were incubated with virus for 1.5 hours at 37˚C and washed three times
in DMEM F12. Organoids were then resuspended in Matrigel and plated as
Time-lapse movies were recorded for 5–7 days during which time the temperature
was held at 37˚C and humidity was held at 5%. Images were acquired every 10
minutes for a duration of 8–24 hours and 10–30 movies were collected in parallel.
Imaris (Bitplane) was used for image analysis. CellTracker was used to label
organoids as described previously (Ewald et al., 2008).
Surface rendering and single-cell tracking were generated using the Imaris
(Bitplane) IsoSurfaces function. Before surface rendering a gaussian smoothing
filter with width of 1 voxel was applied and background signal was subtracted. A
minimum intensity filter and a minimum voxel size filter were manually adjusted
such that the rendered surface visually matched the surface area of individual cells.
IsoSurfaces were tracked using the autoregressive motion algorithm with a
maximum distance of 5 mm and a maximum gap of three frames. The mean cell
speed was calculated as the total track length divided by the time.
We thank Dr. Kent McDonald (UC Berkeley EM Lab) for his advice
and assistance in sample preparation. We thank Chris Booth (Gatan,
Inc.), Joel Mancuso (Gatan, Inc.), and Tom Goddard (UCSF), for
their advice in sample preparation, 3View data collection, and 3D
visualization respectively. We thank Chris Mitchell (JHMI) for
assistance in image processing. We thank Carl Zeiss Microimaging,
The Molecular Foundry at the Lawrence Berkeley Lab, and the
National Center for Electron Microscopy for assistance in pilot 3D
FIB-SEM studies. AJE thanks Paul Matsudaira for suggesting the
possibility that the lateral membrane protrusions represented 3D
This work was supported by funds from the National Cancer Institute
[grant number P50 CA88843]; the National Heart, Lung, and Blood
Foundation Award for Breast Cancer Research to A.J.E.; by the
Director, Office of Science, of the U.S. Department of Energy
[contract DE-AC03-76SF00098] to M.A.; by the University of
California – Berkeley Physical Sciences in Oncology Center to M.A.
and D.M.J.; and the National Cancer Institute and National Institute
of Environmental Health Sciences [grant numbers R01 CA057621,
U01 ES019458] and the California Breast Cancer Research Program
[grant number 17UB-8705] to Z.W.
HL107361]; andthe Safeway
Supplementary material available online at
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