Epithelial-Mesenchymal Transition Stimulates Human Cancer Cells to Extend Microtubule-based Invasive Protrusions and Suppresses Cell Growth in Collagen Gel
Miyazaki K (2012) Epithelial-Mesenchymal Transition Stimulates Human Cancer Cells to Extend Microtubule-
based Invasive Protrusions and Suppresses Cell Growth in Collagen Gel. PLoS ONE 7(12): e53209. doi:10.1371/journal.pone.0053209
Epithelial-Mesenchymal Transition Stimulates Human Cancer Cells to Extend Microtubule-based Invasive Protrusions and Suppresses Cell Growth in Collagen Gel
Jun Oyanagi 0 1
Takashi Ogawa 0 1
Hiroki Sato 0 1
Shouichi Higashi 0 1
Kaoru Miyazaki 0 1
Olivier de Wever, Ghent University, Belgium
0 Current address: Department of Endocrinology, Faculty of Medicine, Kagawa University , Kagawa , Japan
1 1 Graduate School of Integrated Science, Yokohama City University , Yokohama , Japan , 2 Division of Cell Biology, Kihara Institute for Biological Research , Yokohama City Universi, Yokohama , Japan
Epithelial-mesenchymal transition (EMT) is a crucial event in tumor invasion and metastasis. However, most of past EMT studies have been conducted in the conventional two-dimensional (2D) monolayer culture. Therefore, it remains unclear what invasive phenotypes are acquired by EMT-induced cancer cells. To address this point, we attempted to characterize EMT cells in more physiological, three-dimensional (3D) collagen gel culture. EMT was induced by treating three human carcinoma cell lines (A549, Panc-1 and MKN-1) with TGF-. The TGF- treatment stimulated these cells to overexpress the invasion markers laminin c2 and MT1-MMP in 2D culture, in addition to the induction of well-known morphological change and EMT marker expression. EMT induction enhanced cell motility and adhesiveness to fibronectin and collagen in 2D culture. Although EMT cells showed comparable cell growth to control cells in 2D culture, their growth rates were extremely suppressed in soft agar and collagen gel cultures. Most characteristically, EMT-induced cancer cells commonly and markedly extended invasive protrusions in collagen gel. These protrusions were mainly supported by microtubules rather than actin cytoskeleton. Snail-introduced, stable EMT cells showed similar protrusions in 3D conditions without TGF-. Moreover, these protrusions were suppressed by colchicine or inhibitors of heat shock protein 90 (HSP-90) and protein phosphatase 2A. However, MMP inhibitors did not suppress the protrusion formation. These data suggest that EMT enhances tumor cell infiltration into interstitial stroma by extending microtubule-based protrusions and suppressing cell growth. The elevated cell adhesion to fibronectin and collagen and high cell motility also seem important for the tumor invasion.
Funding: This work was supported by Grants-in-Aid (23112517 and 23300351) for Scientific Research from the Ministry of Education, Culture, Sports, Science and
Technology of Japan. (http://www.jsps.go.jp/english/e-grants/index.html) The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Like normal epithelial cells, ductal carcinoma cells in situ
maintain cell polarity, which is supported by cell-cell contact and
cell adhesion to basement membrane. During cancer progression,
some carcinoma cells lose the cell polarity and invade through the
basement membrane and then into connective tissue. This
phenomenon is referred to as epithelial-mesenchymal transition
(EMT) and thought to be a crucial event of cancer progression [1
3]. EMT is critical not only for many developmental steps such as
gastrulation and neural crest formation but also for pathological
events such as wound healing and tissue fibrosis [1,3,4]. EMT is
generally characterized by the loss of epithelial marker E-cadherin,
up-regulation of mesenchymal markers such as N-cadherin and
vimentin, and acquisition of the fibroblast-like spindle cell shape in
monolayer cultures .
EMT of cancer cells is induced typically by TGF- , but
other growth factors and microenvironmental factors, such as
HGF, EGF and FGF, are also able to induce or promote similar
phenotypic changes depending on the cell types [1,3,6]. TGF-
exerts multiple biological activities on the development and the
growth, differentiation, extracellular matrix production and
apoptosis of normal and cancer cells . TGF- is a negative
growth regulator of normal epithelial cells. Whereas TGF-
suppresses tumor cell growth in early stages of carcinogenesis, it
promotes tumor progression in later stages . It has long been
known that TGF- in a combination with other factors such as
TGF-a and EGF, promotes anchorage-independent growth of
normal fibroblasts . The EMT-inducing activity of TGF- is
mainly mediated by the Smad pathway, a major pathway of
complex TGF- signals, which promotes expression of the
EMTrelated transcription factors including Snail, Slug, Twist, and ZEB
1/2 . These transcription factors bind to E-box binding
elements of promoter sequences and suppress expression of
Ecadherin at a transcription level [9,10].
EMT in cancer cells enhances the expression of invasion- or
metastasis-related genes such as matrix metalloproteinases
(MMPs). Our recent study showed that the tumor invasion marker
laminin c2, as well as MMP-9, is induced by the EMT induction
of gastric cancer cells . Other recent studies have suggested
that EMT-induced cancer cells have resistance to anti-cancer
drugs and radioactivity  and have cancer stem cell-like
properties . In spite of a number of past studies on the EMT of
cancer cells, however, it is not clear how EMT contributes to
tumor invasion and metastasis and what invasive phenotypes are
acquired by EMT-induced cancer cells. This is at least in part
owing to the fact that most EMT studies have been performed in
the conventional two-dimensional (2D) culture system. It has
become evident that cells grown in flat 2D culture considerably
differ from those grown in three-dimensional (3D) culture in cell
morphology, proliferation, differentiation, cell-cell interaction,
cell-matrix interaction and gene expression [14,15]. In order to
understand the pathological consequence of EMT of cancer cells,
it seems important to characterize EMT-induced cells in a more
physiological 3D culture system.
In this study, we characterized EMT-induced cancer cells in
both 2D monolayer culture and 3D collagen gel culture, using
three cell lines. We found that EMT-induced cells showed
prominent extension of microtubule-based invasive protrusions
and growth suppression in the 3D collagen gel culture.
EMT Induction of Three Human Cancer Cell Lines
To investigate phenotypic changes induced by EMT of cancer
cells, we used models of three human cancer cell lines. We
previously reported that TGF- and TNF-a synergistically induce
EMT in serum-free culture of MKN-1 human gastric cancer cells
. It has been reported that lung adenocarcinoma cell line A549
 and pancreatic cancer cell line Panc-1  undergo EMT by
treatment with TGF-. In this study, we first tested three cytokines
(TGF-, TNF-a, and EGF) for the EMT induction of A549 and
Panc-1 cells, analyzing expression of some tumor invasion markers
as well as EMT markers. In both cell lines, only TGF- was
required to induce EMT as judged by the mesenchymal
morphological change (Fig. S1), as well as reduction of E-cadherin
expression and induction of vimentin (Fig. 1A and B). TGF- also
simulated the expression of two tumor invasion markers,
MT1MMP and laminin c2 chain, in both cell lines. However,
a combination of TNF-a with TGF- further increased the levels
of the laminin c2 chain and MMP-9 in A549 cells (Fig. 1A). The
laminin c2 chain and MT1-MMP were also induced by
TNFa and/or EGF in Panc-1 cells (Fig. 1B). In addition, we found that
although both TNF-a and TGF- were required for the EMT
induction of MKN-1 cells in serum-free culture, only TGF- was
necessary in the presence of serum (data not shown). These results
indicate that TGF- is the most common and potent inducer of
EMT for the three cancer cell lines, but TNF-a is also required for
efficient expression of some invasion markers, depending on the
Effect of EMT Induction on Cell Adhesion and Migration
in Monolayer Culture
As shown above, TGF- efficiently induced EMT in the three
different carcinoma cell lines. Next, we examined what phenotypes
were acquired by the EMT induction of cancer cells for their
invasive growth. First, the cell adhesion activity of A549 cells was
investigated after EMT induction, using three cell adhesion
substrates, laminin-332, fibronectin and type I collagen. The cells
were pre-treated with TGF- for 24 h and then seeded on these
substrates. As shown in Figs. 2A and 2B, untreated control cells
most efficiently adhered to laminin-332. TGF- treatment only
slightly decreased the cell adhesion to laminin-332. In contrast, it
significantly increased the cell adhesion efficiency to the stromal
substrates fibronectin and type I collagen. The electric time-lapse
cell adhesion assay clearly showed the EMT-induced changes in
the cell adhesion activity of A549 cells to fibronectin and type I
collagen (Fig. 2C). These changes were also observed in Panc-1
and MKN-1 cells (data not shown).
Secondly, cell migration activity was investigated using two
different assays. In in vitro wound healing assay, TGF-
significantly promoted cell migration of A549 and Panc-1 cells in the
absence of TNF-a under serum-supplemented conditions (Fig. 3A
and B). TNF-a did not have additional effect on the cell migration
activity (data not shown). When cell migration was assayed by the
time-lapse video microscopy, the cell motility enhanced by the
TGF- treatment was more clearly shown with the two cell lines
(Fig. 3C). We also confirmed that the TGF- treatment increased
the motility of MKN-1 cells in the wound healing assay (data not
Effect of EMT Induction on Anchorage-dependent and
independent Cell Growth
Thirdly, cell growth activity of EMT-induced cells was
investigated under three different conditions. In 2D monolayer
culture, TGF- treatment did not affect the growth of MKN-1
cells and negligibly or only slightly suppressed that of A549 and
Panc-1 cells (Fig. 4A). When colony formation was examined at
sparse 2D cultures (500 cells/35-mm dish) of A549 and Panc-1
cells, there was no significant difference between the TGF-
treated and non-treated cultures (data not shown). In suspension
culture on non-adhesive plates, the three cell lines slowly grew
forming cell aggregates or spheroids on the plates. Under such
conditions, the TGF- treatment suppressed the growth of
MKN1 cells but had no effect on that of A549 and Panc-1 cells (Fig. 4B).
In contrast, the colony formation of the three cell lines in soft agar
culture was strongly suppressed by the TGF- treatment. Both the
number of total colonies and the total colony area were extremely
reduced in the TGF--treated cell lines than the non-treated ones
(Fig. 4C, 4D, and S2). However, the TGF--treated Panc-1 cells
formed relatively large colonies compared to the non-treated cells
(Fig. S2). The lack of E-cadherin-mediated cell-cell adhesion in
TGF--treated cells might suppress the potentials of cellular
survival and proliferation in the anchorage-independent condition.
Behavior of EMT-induced Cancer Cells in 3D Collagen Gel
Gene expression in vivo is different from that in the monolayer
culture system (15). As a culture system that mimics in vivo
conditions, we used 3D collagen gel culture to further characterize
the EMT-induced cancer cells. Cancer cells were cultured within
collagen gel layer containing TGF- and/or other factors for 7
days. In the 3D collagen gel, MKN-1 cells showed prominent
membrane extensions or protrusions when treated with TGF-
(Fig. 5A and B). A similar morphological change was observed in
A549 and Panc-1 cell lines (Fig. S3). The addition of TNF-a to the
TGF--containing culture further promoted this morphological
change only in the case of MKN-1 cells (Fig. 5B). EGF alone,
which induced EMT in MKN-1 cells, also induced the protrusion
formation in this cell line, but this effect was not observed in A549
and Panc-1 cells. These results suggest that the morphological
change is specific for EMT induction rather than TGF-
We next examined whether EMT induction suppressed cell
growth within collagen gel. As found in soft agar culture, TGF-
strongly suppressed the growth of all cell lines examined in
collagen gel (Fig. 5C). EGF never or only weakly suppressed the
growth of the three cell lines in 3D collagen gel. TNF-a did not
Figure 1. EMT induction in two cancer cell lines. A549 (A) and Panc-1 (B) cells were incubated in serum-free medium with 10 ng/ml EGF, 10 ng/
ml TNF-a, 10 ng/ml TGF-, or TNF-a+TGF-. After 48-h incubation, conditioned media (CMs) and cell lysates were prepared and subjected to
immunoblotting for EMT markers (E-cadherin and vimentin in the cell lysates), the invasion marker laminin c2 (CMs), MT1-MMP (cell lysates), and actin
as the internal loading control (cell lysates). The lowest panels show gelatin zymography of MMP-9 and MMP-2 in the CMs. Values in parentheses
indicate approximate molecular size in kDa. Other experimental conditions are described in Materials and Methods.
have any significant additional effect in these cultures. These data
indicate that the EMT induction promotes prominent extension of
protrusions but suppresses cell growth in the 3D collagen gel
cultures of the three cancer cell lines.
Microtubule-based Structure of EMT-induced Protrusions
in 3D Collagen Gel
To characterize the EMT-induced protrusions, we analyzed
cytoskeletal changes after EMT induction by TGF-. Filamentous
actin (F-actin) and microtubule cytoskeletons of EMT-induced
cells were stained by fluorescence with rhodamine phalloidine and
a tubulin-specific antibody, respectively. MKN-1 cells were
stimulated with TGF- in 2D and 3D cultures and then stained
for the cytoskeletons (Fig.6A and B). In the 2D culture of
EMTinduced cells, robust stress fibers of F-actin, as well as
microtubules, supported the unique cell shape (Fig. 6A). In the 3D
collagen gel, F-actin filaments were strongly detected at the surface
of spheroid structure in unstimulated cells (Fig. 6B). In
TGF-treated cells, peripheral actin filaments were found around the cell
body and protrusions, but stress fibers were rarely found in the
protrusions. In contrast, many bundles of microtubules were
prominently detected in the center of the protrusions in the
EMTinduced cells. In control cells, microtubules were evenly distributed
in the cytoplasm. These patterns of cytoskeletons were commonly
found in MKN-1 and Panc-1 cells (data not shown).
To examine whether microtubule is a principle cytoskeleton
supporting the EMT-induced protrusions, we examined effects
of some cytoskeletal inhibitors on the EMT-induced cell
extension in collagen gel. Cytochalasin B and colchicine are
known to inhibit both polymerization and stabilization of
Factin and microtubules, respectively. When the cells were
treated with these inhibitors at the same time as the TGF-
treatment, the cell extension was partially inhibited by 1 mM
cytochalasin B, but completely by 10 nM colchicine (Fig. 7A).
When these inhibitors were added 24 h after TGF-
stimulation, the protrusions were significantly retracted by colchicine
after 3-h treatment, but not by cytochalasin B (Fig. 7B).
Similarly, two other microtubule inhibitors vinblastin and taxol
(paclitaxel) dose-dependently blocked the cell extension when
added together with TGF- to the 3D culture (Fig. S4). We also
analyzed effects of some signal inhibitors on the EMT-induced
cell extension in collagen gel. The protein phosphatase 2A
(PP2A) inhibitor cantharidin and the heat shock protein-90
(HSP-90) inhibitor radicicol, both which inhibit the stabilization
of microtubules, weakly inhibited the cell extension (Fig. 7C).
When both cantharidin and radicicol were added in
combination, the cell extension was additively suppressed. These data
suggest that the protrusions of EMT-induced cancer cells in 3D
collagen gel are mainly supported by microtubules, and both
PP2A and HSP-90 activities are necessary for the protrusion
formation. Although actin cytoskeleton is required for the
extension of protrusions, it seems unnecessary for maintaining
the protrusion structures.
The protrusions formed in EMT-induced cancer cells within
collagen gel greatly differed in appearance from the cell
morphology in the monolayer culture. This implies that the
interaction of the cells with collagen in the 3D environment is
involved in the protrusion formation. This possibility was
examined by using anti-integrin, neutral antibodies. As expected,
anti-integrin-a2 and anti-integrin-1 antibodies effectively blocked
the extension of protrusions (Fig. 7D). A combination of the two
antibodies more strongly inhibited the protrusion formation than
each antibody. These results demonstrate that the formation of
microtubule-based protrusions requires both EMT-inducing
cytokine and collagen/integrin signals.
Differences from other invasive protrusions. It is well
known that malignant cancer cells invade into 3D extracellular
matrix by extending some kinds of protrusion or projection.
Invadopodium is an actin-based, typical protrusion produced by
invasive cancer cells . MT1-MMP is thought to be a marker of
invadopodia and its activity is required for invadopodium
formation. To test whether the protrusions observed in the
Figure 2. Effect of EMT induction on adhesion activity of A549 cells. (A and B) A ninety-six-well plate was coated with 2 mg/ml laminin-332
(Lm332), 5 mg/ml fibronectin (FN), and 2 mg/ml collagen I (Col I) at 4C overnight, and then blocked with 1.2% BSA for 1 h at 37C. A549 cells were
incubated with 10 ng/ml TGF- in serum-free medium for 24 h. The TGF--treated cells (TGF-) and untreated cells (Control) were suspended and
inoculated onto the plate. After incubation for 30 min, phase contrast images were taken (A) and cell adhesion activity was measured (B). A scale bar
indicates 50 mm. The relative numbers of attached cells were determined as described in Materials and Methods. Each bar indicates the mean 6 SD of
adherent cells in triplicate wells. (C) Electric time-lapse cell adhesion assay with E-plates. The plate was precoated with fibronectin (#, N) and collagen
I (%, &) as shown above, and the adhesion of TGF--treated cells (TGF-: N, &) and untreated cells (Control: #, %) to the plate was monitored every
5 min. Cell index indicates arbitrary unit reflecting attachment and spreading of the cells on microelectrode array in the bottom of the wells. Other
experimental conditions are described in Materials and Methods.
EMT-induced cancer cells inside the collagen gel are identical to
invadopodia, we examined effect of a broad spectrum MMP
inhibitor (TAPI) on the protrusion extension. When TAPI was
simultaneously added with TGF- into the collagen culture, little
or very weak effect on the protrusion extension was obtained in the
three cell lines tested (Fig. S5, AC). Essentially the same results
were obtained when TIMP-2, a natural MMP inhibitor, was used
instead of TAPI (data not shown).
Because Src kinase activity is also associated with the
invadopodium formation, we next examined effects of three kinds
of Src kinase inhibitors, PP1 analog, SU6656 and lavendustin C,
on the protrusion formation in 3D collagen gel. Any kinds of the
inhibitors did not suppress the protrusion formation of MKN-1
cells (Fig. S5, D). These results suggest that the EMT-induced
protrusion is a different cell structure from invadopodium.
Microtubule-based Protrusions in Snail-induced EMT
Cells within Collagen Gel
As shown above, TGF--stimulated cancer cells commonly
extend microtubule-based invasive protrusions in the 3D
collagen gel culture. To more generalize this phenomenon, we
established stably EMT-induced cells by introducing a snail
cDNA expression vector into Panc-1 cells (Snail-Panc-l). Like
TGF--stimulated cells, Snail-Panc-l cells showed scattered cell
morphology as compared with the empty vector-transfected,
control cells (Mock-Panc-1) (Fig. 8A). In accordance with the
morphological change, E-cadherin expression was suppressed
and vimentin expression was enhanced in Snail-Panc-l cells as
compared with the control cells (Fig. 8A). When these
transfected cells were seeded into 3D collagen gel,
Snail-Pancl but not Mock-Panc-1 cells extended microtubule-based, robust
protrusions in the absence of TGF- (Fig. 8B). The extension of
protrusions in Snail-Panc-l cells was strongly inhibited by
colchicine but scarcely by cytochalasin B (Fig. 8C and D).
These data also support that the microtubule-based protrusion
formation reflects EMT in 3D collagen gel.
We also compared the growth potentials in
anchorage-dependent and independent conditions between Mock-Panc-1 and
Snail-Panc-l cells. There was no significant difference in growth
rate between the two kinds of cells in monolayer culture (Fig. 9A).
In soft agar culture, both total colony number and total colony
area were clearly lower in Snail-Panc-l cells than Mock-Panc-1
cells (Fig. 9B and C), but large colonies were more abundant in
Snail-Panc-l cells than the control cells (Fig. 9D). The cell
proliferation in collagen gel was slightly but significantly lower in
Snail-Panc-l cells than the control cells (p,0.05) (Fig. 9E).
Figure 3. Migration activity of EMT-induced cancer cells. (A and B) To measure cell migration activity, A549 (left panels) and Panc-1 (right
panels) cells were subjected to the in vitro wound healing assay in the presence or absence (Control) of TGF-, as described in the text.
Phasecontrast micrographs of the cultures were taken after 16-h incubation. Black broken lines indicate initial wound edges. Scale bar, 50 mm. B) Cell
migration area was estimated by analyzing the pixel with Image J. Each bar indicates the mean 6 SD for the areas of migrated cells in 3 different
fields (right panels). (C) Cells that had been pretreated without or with TGF- for 24 h were incubated in 1% FBS-containing medium without or with
TGF- onto a 24-well plate for 6 h at 37uC. The cell migration was monitored with a time-lapse video system for 12 h. Cell migration distance was
measured for 15 randomly selected cells. Each bar indicates the mean 6 SD for cell migration velocity of 15 cells. Other experimental conditions are
described in Materials and Methods.
In this study, we used EMT models of three human carcinoma
cell lines (A549, Panc-1 and MKN-1) to characterize the
EMTinduced cells in both 2D and 3D culture systems. In agreement
with other studies [16,17], A549 and Panc-1 cells exhibited typical
EMT phenotypes after treatment with TGF- alone in 2D
monolayer cultures. MKN-1 cells required TGF- plus TNF-a in
serum-free medium for EMT induction as reported previously
, but only TGF- was required in serum-containing medium.
Expression of the two invasion markers laminin c2 and
MT1MMP was associated with the EMT induction of these cell lines.
However, expression of MMP-9 and the laminin c2 was much
greater with TGF- plus TNF-a than TGF- alone . These
results suggest that the acquisition of invasive phenotypes of cancer
cells requires TNF-a and/or other factors besides TGF-, even if
TGF- alone is enough for the morphological EMT induction.
The present study revealed that EMT-induced A549 cells more
efficiently adhered to the stromal cell adhesion substrates
fibronectin and type I collagen than uninduced control cells,
though the cell adhesion to the basement membrane substrate
laminin-332 was not changed by the EMT induction. This is
consistent with the fact that expression of stromal extracellular
matrix proteins such as fibronectin and type I collagen is enhanced
by EMT induction . We also confirmed the increased
fibronectin production in the EMT-induced cells in 2D culture
(data not shown). It is highly expected that the EMT-induced
change of cell adhesion activity depends on that of integrin
expression. Furthermore, we showed that the cell migration
potentials of the three cell lines in 2D culture were significantly
Figure 4. Growth of three EMT-induced cancer cell lines in three different conditions. (A) MKN-1 (1.06104 cells), A549 (0.56104 cells) and
Panc-1 (1.06104 cells) were incubated in each well containing 1% FBS-containing medium without (Control) or with TGF- on 24-well culture plates
for 7 days in monolayer culture. After the incubation, the number of cells was measured with a cell counter. Each bar indicates the mean 6 SD of the
cell numbers in triplicate wells. (B) Cells were cultured at a density of 16104 cells per well in 24-well suspension culture plates containing 1%
FBScontaining medium without (Control) or with TGF- for 7 days. The relative number of cells was measured using Dojindo cell counting kit 8. Each bar
indicates the mean 6 SD of the absorbance values at 485 nm in triplicate wells. (C and D) Cells were inoculated at a density of 7,000 cells per 35-mm
dish in 10% FBS-containing soft agar medium without (Control) or with TGF- and incubated for 10 (MKN-1) or 14 (A549 and Panc-1) days. After the
incubation, cells were stained with p-iodonitrotetrazolium violet, and the number of total colonies (C) and total colony area (D) were determined by
Image J and shown as the relative number to the control (100%). Each bar indicates the mean 6 SD in triplicate wells. Other experimental conditions
are described in Materials and Methods.
increased by the EMT induction, in agreement with the results for
Panc-1 cells reported by Horiguchi et al. . These phenotypic
changes, which are consistent with the general concept of EMT,
seem to be required for cancer cell invasion through interstitial
The present study revealed great differences in the phenotypes
of EMT-induced cancer cells between 2D and 3D cultures. Firstly,
the cell growth in response to TGF- was completely different
between the two culture systems. The TGF- treatment did not
have significant effect on the cell growth in monolayer and
suspension cultures of the three cell lines, except for the
suppression of the MKN-1 cell growth in suspension. In contrast,
the growth of the three cell lines in soft agar and collagen gel
cultures was strongly inhibited by the TGF- treatment. TGF- is
a potent growth suppressor of normal epithelial cells and cancer
cells at an earlier stage, whereas it promotes their progression at
a later stage . The canonical TGF--Smad pathway suppresses
cell growth through up-regulating p15 and p21 CDK inhibitors,
but non-smad signal is also involved in the growth suppression
through PP2A activity . However, Ras signal activation
overcomes growth suppressive effect of TGF- .
Moreover, the Ha-Ras-TGF- combination promotes invasive and
Figure 5. Morphological change and growth of EMT-induced MKN-1 cells in 3D collagen gel. (A) MKN-1 cells were incubated in 3D
collagen with or without indicated cytokines on 3-well chamber slides for 7 days. Culture medium was changed every 3rd day. Scale bar, 50 mm. (B)
After 5-day incubation, the numbers of total cell clumps and ones with protrusions were counted in a center field under a microscope, and the
percentage of protrusion-positive cell clumps was calculated. Each bar indicates the mean 6 SD of the relative numbers of protrusion-positive cell
clumps in triplicate wells. (C) After 7-day incubation, the cells in collagen gel were stained with Dojindo cell counting kit 8 for 4 h, and the absorbance
at 485 nm of each culture medium was measured. Each bar indicates the mean 6 SD of the absorbance values in triplicate wells. Other experimental
conditions are described in Materials and Methods.
metastatic behavior of cancer cells [23,24]. A549 and Panc-1 cells
have already acquired a mutation that constitutively activates Ras
[25,26]. MKN-1 cells are also resistant to TGF- because its
receptor expression is very low . These facts may explain the
lack of growth suppression by TGF- in the 2D cultures of these
cell lines. However, constitutive activation of Ras might be
insufficient for escaping from the growth suppression by TGF-
when these cells are placed as single cells into soft agar or collagen
gel. Because E-cadherin-mediated cell-cell adhesion generates
a cell survival signal, the loss of E-cadherin and cell-cell contact by
the EMT induction is likely to reduce the anchorage-independent
growth ability of cells. In addition, it seems reasonable to consider
that invading cancer cells transiently lose their cell proliferation
activity in 3D matrix. However, our results do not necessarily
imply that cancer cells with mesenchymal phenotypes always have
suppressed proliferation activity in 3D conditions. TGF- was
originally found as a cytokine that enhances the
anchorageindependent growth of normal fibroblasts in the presence of
TGFa or EGF (8). Therefore, it seems possible that EMT-induced
cancer cells restore the anchorage-independent growth activity by
the expression or presence of some growth factors and cell
adhesion proteins in vitro and in vivo. It is noted that the stably
EMT-induced cell line Snail-Panc-1 showed relatively high growth
activity in collagen gel as compared with TGF--treated Panc-1
cells. This implies that even if the original cells are identical, the
two types of EMT-induced cells have somewhat different
phenotypes with respect to the growth potential in 3D conditions.
Secondly, we found that the cell morphology of EMT-induced
cells in 3D collagen gel completely differed from that in 2D
monolayer culture. The EMT induction stimulated the three
cancer cell lines to extend prominent invasive protrusions in 3D
collagen gel culture. The protrusion formation was promoted by
TNF-a plus TGF- more strongly than TGF- alone in MKN-1
cells. EGF alone, which is able to induce EMT in MKN-1 cells
, also stimulated the cell extension in this cell line but not the
others. This suggests that the cell extension is associated with EMT
Figure 6. EMT-induced change of cytoskeletal structure of cancer cells cultured in monolayer (2D) and 3D collagen gel cultures.
MKN-1 cells were incubated without (Control) or with 10 ng/ml TGF- in serum-containing medium in 2D culture (A) or 3D collagen gel culture (B) for
3 days. These cells were fluorescence-stained for a-tubulin (green) by using a specific antibody, F-actin by rhodamine phalloidin (red), and DAPI
(blue). Fluorescence images were obtained with a confocal fluorescence microscope. Other experimental conditions are described in Materials and
induction rather than the TGF- activity. The invasive protrusions
were mainly supported by microtubules, and its formation was
blocked by colchicine. Although integrin-dependent actin
cytoskeleton was required for the generation of the invasive
protrusions, it seemed unnecessary for the maintenance of the
protrusions. When mammalian cells receive extracellular stimuli,
they dynamically change their structures, extending pseudopodia
or protrusions. These include filopodia and lamellipodia, both
which are essential structures for cell migration and observed
mostly in 2D cultures . Invadopodium and podosome, which
are thought to be very similar or identical to each other, are well
known as invasive structures of cancer cells [28,18]. All these
structures are actin-based structures. The formation or function of
podosomes and invadopodia require MT1-MMP, Src kinase, PI3
kinase and other signal or cytoskeletal regulators . The cell
protrusion found in this study is a microtubule-based structure,
and its shape and size are clearly different from the above
mentioned cell structures. Furthermore, inhibitors for Src kinase
and PI3K did not inhibit the invasive protrusions in 3D collagen
gel. Therefore, it may be concluded that the EMT-induced
invasive protrusion is a different structure from the four
actinbased cell structures. Recent studies have demonstrated some
similarities between EMT-induced cells and cancer stem cells (12,
13). Like tissue stem cells, cancer stem cells exist in a dormant or
quiescent state in special microenvironments called niches .
This is consistent with our finding that EMT-induced cells have
suppressed growth activity in 3D collagen gel. However, it is
unlikely that cancer stem cells actively migrate through 3D matrix,
extending protrusions. There seem to be important differences
between EMT-induced cancer cells and cancer stem cells.
Figure 7. Effects of various inhibitors on protrusion formation of MKN-1 cells in 3D collagen gel. MKN-1 cells were incubated with
10 ng/ml TGF- in serum-containing medium in 3D collagen gel culture, as described in Figures 5 and 6. To the collagen culture were added various
inhibitors, and the protrusion formation was quantified 24 h later (A, C and D) or 3 h later (B). (A) The indicated concentrations of cytochalasin B and
colchicine were added into the culture medium at the same time as the TGF- addition. (B) Cytochalasin B (5 mM) and colchicine (1 mM) were added
into the culture medium after incubation with TGF- for 24 h. (C) MKN-1 cells were treated without (Control) or with cantharidin (1 mM) and/or
radicicol (1 mM) as described in (A). (D) MKN-1 cells were pretreated with 10 mg/ml of each neutral antibody at 37C for 30 min, and the pretreated
cells were embedded into collagen gel containing 10 mg/ml of the indicated anti-integrin antibody and 10 ng/ml TGF-. All these inhibitors were not
cytotoxic at least under the above experimental conditions, as analyzed by the trypan blue staining. However, cytotoxic effects became evident when
the cells were incubated with 5 mM cytochalasin B or 1 mM colchicine for 24 h.
Recently, Whipple et al. [33,34] reported a novel
microtubulebased protrusion microtentacle, which is induced in suspension
by EMT. The microtubule-associated protein tau is essential for
the microtentacle formation . In our study, PP2A and
HSP90, both which regulate microtubule stability by acting on tau
[36,37], were also essential for the protrusion formation in 3D
collagen gel. The invasive protrusions in this study required
integrin-mediated interaction with collagen and greatly differed
from microtentacles in appearance. Many recent studies have
investigated the mechanism of cell invasion within 3D collagen gel
or 3D matrix. For example, invasive human cancer cells such as
HT1080 fibrosarcoma and MDA-MB-231 breast carcinoma cells
invade through collagen gel by projecting a long membrane
extension [38,39]. The long protrusions of MDA-MB-231 cells are
supported by microtubules . Although the mesenchymal cell
invasion requires MMP and Src activities, the presence of MMP
inhibitors allows them to migrate through the collagen matrix by
exhibiting an amoeboid cell structure . However, the MMP
requirement may depend on the cross-linkage of collagen fibers
used in experiments . It is also well known that when MDCK
cells are treated with HGF in collagen gel, they start to invade the
collagen gel, generating a membrane extension . A recent
study has shown that the invasive structure of MDCK cells is
supported by microtubules . Similarly, Ha-Ras-transformed
mammary epithelial cells form tubular structures in collagen gel by
TGF- treatment . These invasive structures seem to be very
close or essentially identical to the invasive protrusions of the
EMT-induced cancer cells found in the present study. It has been
reported that signal mediators for actin cytoskeleton, e.g.
PI3KAkt signal  and Rho-GTPases [44,45], also regulate
microtubule stability, and TGF- activates Rho and Rac signals [46,47].
In the present study, however, neither PI3K-Akt signal inhibitors
nor a ROCK inhibitor suppressed the protrusion formation.
These discrepancies suggest substantial differences in cytoskeletal
regulation between 2D and 3D conditions. Further studies are
required for clarifying how TGF- induces the microtubule-based
invasive protrusions in 3D collagen gel.
In conclusion, our study demonstrates that EMT-induced
cancer cells acquire a mesenchymal phenotype and invade into
collagen matrix by projecting robust microtubule-based
protrusions. EMT cells lose cell proliferation potential in collagen matrix.
This suggests that mesenchymal-epithelial transition (MET) under
a different microenvironment is required for the cancer cells to
grow again. Our findings provide a clinical clue to suppress tumor
invasion in vivo.
Materials and Methods
Antibodies and Reagents
Mouse monoclonal antibody (mAb) against the human laminin
c2 chain (D4B5) was established and characterized previously
. Other antibodies used and their source are as follows: mouse
mAb against human E-cadherin from Becton Dickinson (Franklin
Lakes, NJ), mouse mAb against human vimentin from Sigma
Aldrich (St. Louis, MO); mouse mAb against human a-tubulin and
function-blocking antibodies against integrins a2 (P1E6), a5
(P1D6) and 1 (6S6) from Chemicon (Temecula, CA); rabbit
polyclonal antibody against human actin from Biomedical
Technologies (Stoughton, MA), and FITC-conjugated anti-mouse
IgG antibody from Vector laboratories (Burlingame, CA). Human
transforming growth factor-1 (TGF-1), human epidermal
growth factor (EGF), human tumor necrosis factor-a (TNF-a),
colchicine, cytochalasin B, and SB431542 were purchased from
Wako pure chemical (Osaka, Japan), and radicicol and cantharidin
from Calbiochem (La Jolla, CA).
Cell Culture and Preparation of Conditioned Media and
Human adenocarcinoma cell lines of the stomach (MKN-1) and
lung (A549) were provided from Japanese Collection of Research
and Bioresources (JCRB, Tokyo, Japan) in 1993 and stored in
liquid nitrogen after a few passages . MKN-1 and A549 cells
secrete laminin-332 and laminin-511, respectively. Their laminin
Figure 9. Growth of Snail-expressed Panc-1 cells in three different conditions. (A) Monolayer culture. Mock-Panc-1 (open column) and
Snail-Panc-1 (closed column) were seeded at a density of 1.06104 cells per well of 24-well plates in 10% FBS-containing medium and incubated for 7
days. After the incubation, the number of cells was measured with a cell counter. (BD) Soft agar culture. Mock-Panc-1 and Snail-Panc-1 cells were
cultured in soft agar medium for 14 days. After the incubation, the number of total colonies (B) and total colony area (C) were analyzed by Image J.
The soft agar cultures were photographed and each typical image is shown in (D). Scale bar, 500 mm. Other experimental conditions were the same
as described in Figure 4. (E) Collagen culture. Mock-Panc-1 and Snail-Panc-1 cells were cultured in 3D collagen gel for 7 days as described in Figure 5.
After the incubation, the relative number of cells was measured using Dojindo cell counting kit 8.
production was confirmed in this study. Pancreatic
adenocarcinoma cell line Panc-1 was provided from JCRB in 2004. Its identity
was confirmed in this study from similarity in the response to
TGF- to that in a previous study . These cell lines were
cultured in DMEM/F12 medium (Invitrogen, Carlsbed, CA)
supplemented with 10% fetal bovine serum (FBS) (Nichirei
Biosciences, Tokyo) at 37C in a humidified atmosphere of 5%
CO2 and 95% air. This medium was used as the standard
serumcontaining medium unless otherwise noted. Conditioned media
were prepared as reported previously . Cells were grown to
confluence in serum-containing medium. After the incubation, the
cultures were further incubated in serum-free medium with or
without cytokines for 2 days. The resultant conditioned media
were collected, dialyzed against distilled water and lyophilized.
The dried protein was dissolved in a 1/50 volume of distilled
water. Cells were lysed in RIPA buffer (20 mM Tris-HCl, pH7.5,
150 mM NaCl, 5 mM EDTA, 2.5 mM tetra-sodium
pyrophosphate, 1 mM sodium orthovanadate, 10 mM NaF, a protease
inhibitor mixture (Wako), 1% Nonidet P-40, 0.5% sodium
deoxycholate, and 0.1% SDS) and centrifuged at 10,0006g for
10 min. The resultant supernatants were used as cell lysates.
SDS-PAGE was performed on 6 or 10% polyacrylamide gels
under non-reducing or reducing conditions. Immunoblotting of
EMT marker proteins and gelatin zymography of MMPs were
carried out as reported previously .
Cell Adhesion Assays
Cells were incubated at a density of 2.06104 cells per well in
0.1 ml serum-free medium for 30 min at 37C on 96-well
microtiter plates, which had been precoated with each cell
adhesion substrate and blocked with 1.2% (w/v) bovine serum
albumin (BSA). After non-adherent cells were removed by gentle
agitation, adherent cells were fixed with 2.5% glutaraldehyde and
stained with 0.5% (w/v) crystalviolet for 30 min at room
temperature. Each well was measured for absorbance at 595 nm
with Plate Chameleon V (Hydex; Turka, Finland). Alternatively,
time-lapse cell adhesion was electrically analyzed using the
xCELLigence System Real-Time Cell Analyzer (Roche; Basel,
Switzerland), according to the manufactures instruction manual.
E-plate was coated with each substrate protein and blocked with
BSA. Cell attachment was measured every 5 min.
Cell Migration Assays
Confluent cultures were incubated with 10 ng/ml TGF- in
serum-containing medium for 24 h and then with 10 mg/ml
mitomycin C for 2 h to prevent cell growth. The cell monolayer
was scratched with a 200 ml pipette tip, washed with PBS, and
incubated in fresh DMEM/F12 plus 1% FBS with or without
TGF- for 16 h. Phase-contrast micrographs were taken
immediately and 16 h after wounding. The scratched area was analyzed
by Image J. In random cell migration assay, cells stimulated by
TGF- for 24 h were inoculated in 1% FBS-supplemented
medium with or without TGF-. After 6-h incubation, cell
migration was monitored at 37C with a time-lapse video system
for 12 h. The distance of cell migration was measured by using
a video monitor (Olympus VM-30; Tokyo).
Cell Growth Assays
For the assay in monolayer culture, cells were incubated on
24well culture plates in DMEM/F12 plus 1% FBS supplemented
with or without 10 ng/ml TGF-. After incubation for 7 days,
grown cells were harvested and counted with a cell counter
(Sysmex CDA-500; Hyogo, Japan). For suspension culture, cells
were incubated in the same medium as above on 24-well Celltight
X suspension culture plates (Sumilon; Tokyo). After incubation for
7 days, the relative number of grown cells was determined with the
cell counting kit 8 (Dojindo; Kumamoto, Japan). For soft agar
culture, cells were incubated in 0.33% (w/v) agar medium
containing 10% FBS with or without TGF-, which was overlying
1% (w/v) agar layer in 35-mm dishes. The cultures were incubated
at 37C for 10 or 14 days. Colonies were visualized by incubating
with 0.5 mg/ml of p-iodonitrotetrazolium violet for 24 h, and the
total number and area of colonies in a center field were analyzed
by Image J.
Collagen Gel Culture
Collagen gel culture was performed using bovine type I collagen
(Nitta Gelatin, Osaka, Japan) on 3-well assay slides (AR Brown,
Tokyo, Japan). A base layer of 2.1 mg/ml collagen in
serumcontaining medium (30 ml) was prepared in each well. Thirty ml of
cell suspension containing 15,000 cells was mixed with 120 ml of
the unsolidified collagen gel solution, and its 30-ml aliquot was
placed on the base layer. After 30-min incubation at 37C, the
upper gel was overlaid with 100 ml of serum-containing medium
with or without growth factors, and incubated for 1 week. The
medium was replaced every 3rd day. Cell growth was assessed
using the cell counting kit 8.
Immunofluorescent Staining of Cells in Collagen Gel
Cells cultured in collagen gel for 3 days were fixed in 10%
formalin in PBS for 1 h and washed 3 times with PBS. The cells
were permeabilized with 0.1% (v/v) Triton X-100 in PBS (PBST)
for 1 h, blocked with 5% BSA in PBS for 2 h, and incubated
overnight at 4 C with the primary antibody against a-tubulin
diluted with 5% BSA in Triton X-100/PBS. The cells were then
stained with FITC-labeled secondary antibody against mouse IgG,
rhodamine phalloidin, and DAPI for 3 h at room temperature.
The fluorescence image was observed using a confocal
fluorescence microscope (Olympus FV1000-D BX61; Tokyo).
Construction of Snail Expression Vector and Transfection
Snail cDNA clone (pF1KB7546) was purchased from Kazusa
DNA research institute (Chiba, Japan). pF1KB7546 was digested
with SpeI and SmaI, and cloned into a Xba-SmaI site of the
mammalian expression vector pBOS-CITE-NEO to make
pBOSCITE-NEO-snail . Panc-1 cells were transfected with an
empty vector (Mock-Panc-1) or the snail expression vector
(SnailPanc-1) using Xtreme gene 9 transfection reagents (Roche, Basel,
Switzerland), and their stable transfectants were selected with
500 mg/ml of G418 (Calbiochem; La Jolla, CA).
Figure S1 EMT induction in two cancer cell lines, A549
and Panc-1. A549 and Panc-1 cells were incubated with 10 ng/
ml EGF, 10 ng/ml TNF-a, 10 ng/ml TGF-, or TNF-a+TGF-
in serum-free medium. After incubation for 48 h, phase contrast
micrographs were taken. x 300.
Figure S2 Colony formation of three cell lines in soft
agar cultures with or without TGF-. MKN-1, A549, and
Panc-1 cells were incubated in soft agar medium with or without
TGF-. After incubation for 14 days, the cultures were
photographed. Each typical image is shown. Other experimental
conditions were the same as described in Fig. 4C.
Figure S4 Effects of paclitaxel and vinblastin on
protrusion formation of MKN-1 cells in 3D collagen gel.
MKN-1 cells were incubated without (open columns) or with the
indicated concentrations of paclitaxel (A) or vinblastin (B) 10 mM
TAPI-1 in the presence of TGF- for 5 days, and protrusion
formation was quantified. Other experimental conditions are
described in Figures 5 and 7.
Figure S5 Effects of synthetic MMP inhibitor and signal
inhibitors on protrusion formation of MKN-1 cells in 3D
collagen gel. MKN-1(A), A549 (B), and Panc-1 (C) cells were
incubated without (open columns) or with 10 mM TAPI-1 in the
absence (Control) or presence (TGF-) of TGF- for 5 days, and
protrusion formation was quantified. (D) MKN-1 cells were
incubated without (None) or with the following inhibitors in the
absence (open column) or presence (closed columns) of TGF- for
24 h: PP1-analog (1 mM), SU6656 (1 mM), or lavendustin C
(1 mM). Other experimental conditions are described in Figures 5
Conceived and designed the experiments: JO SH KM. Performed the
experiments: JO TO HS. Analyzed the data: JO SH KM. Contributed
reagents/materials/analysis tools: JO TO. Wrote the paper: JO KM.
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