Cell motility is essential for many aspects of metastasis; however,
few molecular markers exist that can predict the migratory potential
of a tumor cell in vivo. Intravital multiphoton imaging in animal
models can be used to characterize carcinoma and stromal cell
behavior within intact primary tumors in detail (Condeelis and
Segall, 2003; Wang et al., 2007; Egeblad et al., 2008; Kedrin et al.,
2008; Andresen et al., 2009; Perentes et al., 2009). Such imaging
approaches yield direct information at single-cell resolution and
permit quantification of cell motility, interactions between tumor
and stromal cells, and direct observation of invasion, intravasation
and extravasation. In mammary tumors, this technology was used
to describe the microenvironments in which tumor cells invade,
migrate and intravasate, and revealed essential roles for
macrophages in these events (reviewed in Condeelis and Segall,
2003; Condeelis and Pollard, 2006; Yamaguchi et al., 2006; Kedrin
et al., 2007). In particular, chemotaxis of tumor cells toward
macrophages is essential for invasion in mouse primary mammary
tumors (Wyckoff et al., 2004; Goswami et al., 2005), whereas
chemotaxis of tumor cells toward peri-vascular macrophages is
essential for intravasation (Wyckoff et al., 2007).
Expression profiling of invasive tumor cells captured from the
primary tumor was used to obtain molecular information regarding
the pathways mediating carcinoma cell invasion and intravasation
(Wyckoff et al., 2000a; Wang et al., 2004; Wang et al., 2007). The
‘invasion signature’ obtained from this profile revealed sets of
coordinated expression changes associated with increased invasive
potential (Goswami et al., 2004; Wang et al., 2004; Wang et al.,
2006; Wang et al., 2007; Goswami et al., 2009). Mena, a regulator
of actin polymerization and cell migration, is upregulated in
invasive tumor cells obtained from rat, mouse and human tumors
(Di Modugno et al., 2006; Goswami et al., 2009; Robinson et al.,
2009). Conservation of Mena upregulation in invasive tumor cells
across species suggests that it plays a crucial role in metastatic
In patients, Mena expression correlates with metastatic risk:
relatively high Mena expression has been observed in patient
samples from high-risk primary and metastatic breast tumors (Di
Modugno et al., 2006), as well as cervical, colorectal and pancreatic
cancers compared with low-risk cases (Gurzu et al., 2008; Pino et
al., 2008; Gurzu et al., 2009). Mena is also a component of a
marker for metastatic risk called TMEM (tumor micro-environment
for metastasis) (Robinson et al., 2009). TMEMs are identified by
co-localization of Mena-positive tumor cells, macrophages and
endothelial cells, and the TMEM score predicts risk independently
of clinical subtype of cancer (Robinson et al., 2009). Therefore, the
contribution of Mena to metastasis is independent of clinical
These findings emphasize the importance of determining the
mechanism by which Mena and its isoforms differentially affect
We have shown previously that distinct Mena isoforms are expressed in invasive and migratory tumor cells in vivo and that the
invasion isoform (MenaINV) potentiates carcinoma cell metastasis in murine models of breast cancer. However, the specific step of
metastatic progression affected by this isoform and the effects on metastasis of the Mena11a isoform, expressed in primary tumor cells,
are largely unknown. Here, we provide evidence that elevated MenaINVincreases coordinated streaming motility, and enhances
transendothelial migration and intravasation of tumor cells. We demonstrate that promotion of these early stages of metastasis by
MenaINVis dependent on a macrophage–tumor cell paracrine loop. Our studies also show that increased Mena11a expression correlates
with decreased expression of colony-stimulating factor 1 and a dramatically decreased ability to participate in paracrine-mediated
invasion and intravasation. Our results illustrate the importance of paracrine-mediated cell streaming and intravasation on tumor cell
dissemination, and demonstrate that the relative abundance of MenaINVand Mena11a helps to regulate these key stages of metastatic
progression in breast cancer cells.
Key words: Breast cancer, Cell motility, Intravital imaging, Metastasis, Transendothelial migration
Accepted 18 February 2011
Journal of Cell Science 124, 2120-2131
© 2011. Published by The Company of Biologists Ltd
Mena invasive (MenaINV) promotes multicellular
streaming motility and transendothelial migration in a
mouse model of breast cancer
Evanthia T. Roussos1,*, Michele Balsamo2, Shannon K. Alford2, Jeffrey B. Wyckoff1,3, Bojana Gligorijevic1,
Yarong Wang1, Maria Pozzuto4, Robert Stobezki5, Sumanta Goswami1,5, Jeffrey E. Segall1,
Douglas A. Lauffenburger2, Anne R. Bresnick4, Frank B. Gertler2,* and John S. Condeelis1,3,*
1Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
2David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
3Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA
4Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA
5Department of Biology, Yeshiva University, New York, NY 10033, USA
*Authors for correspondence (firstname.lastname@example.org; email@example.com; firstname.lastname@example.org)
Journal of Cell Science
metastatic progression. Mena is a member of the Ena/VASP family
of proteins and binds actin to regulate the geometry and assembly
of filament networks through: (1) an anti-capping protein activity
(Bear et al., 2002; Barzik et al., 2005; Hansen and Mullins, 2010)
that involves binding to profilin and both G- and F-actin; (2) Mena
tetramerization, and (3) reduction in the density of actin-related
proteins 2 and 3 (Arp2/3)-mediated branching (Gertler et al., 1996;
Barzik et al., 2005; Ferron et al., 2007; Pasic et al., 2008; Bear and
Gertler, 2009; Hansen and Mullins, 2010). Alternative splicing for
the Mena gene has been reported: a 19 amino acid residue insertion
just after the EVH1 domain generates the Mena invasion isoform
(MenaINV, formerly Mena+++) (Gertler et al., 1996; Philippar et al.,
2008), whereas a 21 residue insertion in the EVH2 domain
generates the Mena11a isoform (Di Modugno et al., 2007). A
comparison of the invasive and migratory tumor cells collected in
vivo, with primary tumor cells isolated from mouse, rat and human
cell-line-derived mammary tumors, revealed that MenaINV
expression is upregulated and Mena11a is downregulated selectively
in the invasive and migrating carcinoma cell population (Goswami
et al., 2009). The differential regulation of Mena isoforms across
species suggests that these two isoforms have important roles in
invasion and metastasis.
In previous studies, we showed that expression of MenaINVin a
xenograft mouse mammary tumor promotes increased formation of
spontaneous lung metastases from orthotopic tumors and alters the
sensitivity of tumor cells to epidermal growth factor (EGF) (Philippar
et al., 2008). We undertook the current study to identify the step(s)
in the metastatic cascade that are affected by MenaINVexpression
and investigate the effect of expression of the second regulated
isoform, Mena11a, on metastatic progression. In particular, we
dissected each step of metastatic progression to determine which
steps are affected by expression of MenaINVthat ultimately leads to
enhancement of metastatic dissemination, and whether these same
steps are also affected by Mena11a expression in tumor cells.
We chose MTLn3 cells for our studies because they are well
characterized with respect to tumor cell invasion, migration and
metastasis (Levea et al., 2000; Sahai, 2005; Le Devedec et al.,
2009; Le Devedec et al., 2010), tumor–stromal cell interactions
(Sahai, 2005), TGF signaling in metastatic progression (Giampieri
et al., 2009), and the role of Mena in breast cancer metastasis
(Philippar et al., 2008; Goswami et al., 2009). MTLn3 cells are
derived from the clonal selection of metastatic lung lesions from
rats with mammary tumors (Neri et al., 1982). These rat mammary
tumors have been characterized as estrogen-independent and they
metastasize to the lymph nodes and lungs (Neri et al., 1982).
Evaluation of vimentin and cytokeratins in MTLn3 mammary
tumors, associated lymph nodes and lung metastases revealed that
MTLn3 tumor cells are comparable to a basal-like subtype of
breast cancer (Lichtner et al., 1989).
MenaINVpromotes coordinated cell migration in vivo in the
form of streams of single cells
Previously, we found that expression of Mena and MenaINV
increases in vivo cell motility, which we hypothesized contributes
to the increased lung metastasis observed with these cells (Philippar
et al., 2008). Different types of motility are thought to play diverse
roles during tumor cell invasion (Wolf et al., 2003; Gaggioli et al.,
2007; Ilina and Friedl, 2009; Friedl and Wolf, 2010), therefore we
hypothesized that MenaINVexpression promotes a type of motility
that supports enhanced tumor cell invasion. To address this
Mena isoforms in migration and intravasation
hypothesis, we used multiphoton-based intravital imaging (IVI) to
examine the types of motility displayed by tumor cells expressing
the different Mena isoforms. In all tumors, we generally observed
two patterns of movement: coordinated cell movement whereby
the cells align and move in an ordered single file line (streaming)
(Fig. 1A, top panel; supplementary material Movie 1), and random
cellular movement whereby carcinoma cells move independently
of other carcinoma cells in an uncoordinated manner (Fig. 1A,
bottom panel; supplementary material Movie 2). Both MenaINV-
expressing and Mena11a-expressing tumor cells exhibited streaming
and random movement in vivo. However, streaming movement
was significantly more common in MenaINV-expressing tumor cells
in vivo (Fig. 1B). Quantification of cells moving within the primary
tumor showed that MenaINVexpression significantly increased
both random and streaming tumor cell movement compared with
that seen with GFP-expressing control cells and Mena11a-
expressing primary mammary tumors. Both movement types were
only slightly increased in Mena11a-expressing tumor cells (Fig.
1B). To characterize streaming motility further, time-lapse images
were analyzed for cell crawling (supplementary material Fig. S1A),
and we used photoconversion from green to red to measure the
stability of the streams over a 24-hour period (Kedrin et al., 2008).
Carcinoma cell streams were observed over 24 hours after
photoconversion, suggesting that streaming is a long-lived behavior
(Fig. 1C) involving crawling cells (supplementary material Fig.
S1A). Results from co-injection of MTLn3 cells expressing GFP–
Mena11a–GFP or Cerulean–MenaINVshowed that the tissue
architecture specific to each Mena isoform type is preserved as
compared with injection of either Mena11a- or MenaINV-expressing
cells alone (data not shown).
Streaming cell movement is more productive than random
We studied the underlying motility parameters contributing to
streaming and random movement by determining cell speed, net
path length, directionality and turning frequency in vivo. In vivo,
streaming cells moved significantly faster than randomly moving
cells, regardless of Mena isoform expression (supplementary
material Fig. S1B), with average speeds of greater than 1.9
m/minute. All cell types participating in random movement
exhibited a narrow distribution of velocities (supplementary
material Fig. S1Ci), whereas cells that participated in streaming
movement had a broad distribution of velocities (supplementary
material Fig. S1Cii). This suggests that random cell movement is
autonomous, whereas streaming cells exhibit velocities dependent
on multiple cell–cell signaling interactions.
The directionality, net path length and turning frequency of a
cell are measures of cell locomotion efficiency. In general, the net
path length (supplementary material Fig. S1D) and directionality
(supplementary material Fig. S1E) of streaming carcinoma cells in
vivo were increased, whereas turning frequency was decreased
(supplementary material Fig. S1F) compared with randomly moving
cells. These results indicate that streaming cell movement is more
efficient because cells that stream move faster, farther and turn less
frequently. The increased streaming observed in vivo for cells
expressing MenaINV(supplementary material Fig. S1B) means that
MenaINV-expressing cells move more efficiently in vivo. In steady-
state conditions in vitro, cells only move randomly and the Mena
isoform-expressing cells did not differ significantly from each
other in speed and directionality. This suggests that additional
factors are required for streaming that are not present in vitro
Journal of Cell Science
(supplementary material Fig. S2A,B). We investigated these
additional in vivo factors and outline the results below.
In vivo invasion is enhanced by the expression of MenaINV
and suppressed by the expression of Mena11a
To determine whether the enhanced streaming exhibited by
MenaINV-expressing carcinoma cells correlates with chemotaxis-
dependent invasion, we used the in vivo invasion assay to evaluate
the ability of carcinoma cells to invade towards EGF in vivo
(Wyckoff et al., 2000a). We showed previously that MTLn3 cells
exhibit a characteristic biphasic response to EGF whereby maximal
chemotactic invasion is achieved in response to 25nM EGF (Segall
et al., 1996; Wyckoff et al., 2000a). Expression of MenaINVshifts
this biphasic response, and maximal invasion is achieved in
response to 1nM EGF (Fig. 2A) (Philippar et al., 2008). This result
demonstrates that sensitivity to EGF chemotaxis is increased in
vivo and is consistent with the increased sensitivity of MenaINV-
expressing cells to EGF in vitro (supplementary material Fig.
S3A,B) (Philippar et al., 2008). In vitro, MenaINV-expressing cells
exhibit protrusive activity in response to EGF concentrations as
low as 0.1 nM, whereas cells expressing GFP and Mena11a did
not respond to stimulation with either 0.1 or 0.5 nM EGF
2122Journal of Cell Science 124 (13)
(supplementary material Fig. S3A,B). Importantly, MenaINV
expression not only sensitizes tumor cells to EGF, but also
significantly increases the number of invasive cells collected with
the peak concentration of EGF, which indicates more efficient cell
migration (Fig. 2A). Mena11a-expressing tumors did not invade
significantly above background levels in response to a broad range
of EGF concentration (Fig. 2A). Thus, Mena11a and MenaINV
have opposite effects on chemotaxis-dependent invasion in vivo.
Expression of Mena isoforms alters paracrine loop
signaling with macrophages during in vivo invasion
Using the in vivo invasion assay, tumor cells have been observed
to chemotax into needles containing either EGF or colony-
stimulating factor 1 (CSF1) (Wyckoff et al., 2004; Patsialou et al.,
2009). This can only occur if a paracrine signaling relay is
established with macrophages because, in the absence of
macrophages, the chemotatic signal cannot be transmitted over
long distances and few cells are collected (Wyckoff et al., 2004).
Therefore, we looked at whether the tumor cell–macrophage
paracrine loop is involved in the enhanced in vivo invasion of
MenaINV-expressing cells and the suppression of invasion in
Mena11a-expressing cells. Both macrophages and tumor cells enter
Fig. 1. MenaINVpromotes coordinated carcinoma cell streaming within
the primary tumor significantly more than other Mena isoforms.
(A)Multiphoton microscopy images (20?) of MenaINV- and Mena11a-
expressing mammary tumors in mice. Upper panels illustrate MTLn3–
Cerulean–EGFP–MenaINVcells at different intervals in time, moving in a
stream; cells outlined in white follow the same path (direction indicated
with white arrows in far left panel) as they move through the tumor. Lower
panels illustrate MTLn3–Cerulean–EGFP–Mena11a cells at different
intervals of time, moving randomly; cells outlined in white are moving in
different directions from each other (directions indicated with white arrows
in far left and middle panels). Scale bars: 25m. Green, Cerulean MTLn3
cells expressing either EGFP–MenaINVor EGFP–Mena11a. Purple,
collagen I second harmonic. Vector diagrams to the right illustrate
movement patterns of streaming (top vector diagram) versus randomly
moving (bottom vector diagram) cells in the panels to the left. Vector
diagrams are representative of all cell types participating in either
streaming or random movement. (B)Average number of tumors cells
moving randomly (white bars) or streaming (gray bars) per field quantified
from IVI of primary mammary tumors derived from mammary gland
injection of cell types indicated; 30–50 fields analyzed per condition. P
values are indicated above bars. Error bars indicate s.e.m. (C)Multiphoton
microscopy of MenaINV-expressing MTLn3 tumor cells coexpressing
Dendra2 moving coordinately in a cell stream. Images taken at 20? at
time 0 (image taken immediately following Dendra2 photoconversion) and
24 hours following Dendra2 photoconversion from green to red. Red area
results from the same red photoconverted tumor cells in both images.
White arrow, Denra2 photoconverted carcinoma cells in a stream. Green,
Dendra2 in MTLn3–MenaINVcells. Scale bars: 50m.
Journal of Cell Science
collection needles during in vivo invasion (Wyckoff et al., 2004),
and typing of cells collected following in vivo invasion confirmed
the presence of both tumor cells and macrophages in collections
from tumors expressing GFP, MenaINVand Mena11a (data not
To assess the extent of paracrine signaling between
macrophages and MenaINV-expressing carcinoma cells during in
vivo invasion, we performed the in vivo invasion assay in the
presence of either 6.25 nM Erlotinib (Tarceva), an EGF receptor
(EGFR) tyrosine kinase inhibitor, or a mouse CSF1 receptor-
blocking antibody (-CSF1R) (Wyckoff et al., 2004). In vivo
invasion of both MenaINV- and GFP-expressing cells was reduced
to background levels in assays with Erlotinib as compared with
Mena isoforms in migration and intravasation
invasion toward needles containing EGF+DMSO or EGF alone
(Fig. 2B), demonstrating the requirement for EGFR-mediated
stimulation for invasion. Similarly, invasion of both MenaINV-
and GFP-expressing cells decreased significantly with needles
containing -CSF1R as compared with invasion toward needles
containing EGF+DMSO or EGF+IgG antibodies (control IgG)
(Fig. 2C), thus suggesting the necessity of EGF production and
signal propagation by macrophages. These results are consistent
with the requirement for co-migrating macrophages in tumor cell
migration, as shown in Fig. 3D and discussed below. Together,
these results demonstrate the requirement for paracrine signaling
between MenaINV-expressing tumor cells and macrophages in
Fig. 2. MenaINVand Mena11a have
opposite effects on invasion in vivo and in
vitro. (A)In vivo invasion assay: EGF dose-
response curve of cells collected from
primary mammary tumors derived from
mammary gland injection of indicated cell
types. Values represent averages of 15–25
needles per xenograft model. (B)In vivo
invasion in the presence of Erlotinib. Cells
were collected from mammary tumors
derived from mammary gland injection of
each cell type. Bars represent contents of
15–25 needles per xenograft model. (C)In
vivo invasion in the presence of CSF1R-
blocking antibody (-CSF1R): Cells
collected from mammary tumors derived
from mammary gland injection of each cell
type. Bars represent contents of 15–25
needles per xenograft model. (D)Real time
PCR of cell lines for expression of CSF1.
Each bar represents n3 for three
independent experiments. (E)In vitro 3D
invasion assay. Proportion of GFP- and
Mena11a-expressing cells invading collagen
in the absence (white bars) or presence
(gray bars) of macrophages. Each bar
represents n3. P values are indicated above
bars. Error bars indicate s.e.m.
Journal of Cell Science
Both CSF1 secretion and EGF binding to the EGFR by
carcinoma cells are essential components of the carcinoma cell–
macrophage paracrine loop (Wyckoff et al., 2004; Patsialou et al.,
2009). We used real time PCR to examine the relative mRNA
expression of CSF1 and EGFR in the Mena isoform-expressing
cells lines in culture to determine whether changes in the expression
of these signaling molecules could contribute to the observed
differences in EGF-dependent in vivo invasion (Wyckoff et al.,
2004). Mena11a-expressing cells showed a fourfold decrease in
CSF1 expression as compared with GFP-expressing cells, whereas
CSF1 expression in Mena- and MenaINV-expressing cells was
unchanged (Fig. 2D). We previously reported no difference in
EGFR expression in cells expressing either Mena or MenaINVas
compared with MTLn3 cells (Philippar et al., 2008). Cells
expressing Mena11a also showed no statistical difference in
expression of EGFR compared with GFP- and MenaINV-expressing
cells (supplementary material Fig. S4), indicating that altered
receptor levels do not contribute to the altered EGF-dependent
phenotypes observed in the different cell types. Thus, the inability
of Mena11a-expressing cells to participate in macrophage-
dependent invasion might arise in part due decreased CSF1
expression along with a reduction in direct response EGF (Fig.
2124Journal of Cell Science 124 (13)
To determine whether suppression of chemotaxis-dependent
invasion of Mena11a-expressing tumor cells resulted from
differences in the ability of these tumor cells to co-migrate with
macrophages, we used a 3D invasion assay that measures
macrophage-dependent co-migration of carcinoma cells with
macrophages in collagen (Goswami et al., 2005). Although addition
of macrophages to GFP-expressing tumor cells significantly
increased 3D invasion, addition of macrophages to Mena11a-
expressing cells did not significantly increase invasion (Fig. 2E).
This is consistent with the reduced response to EGF and the
reduced CSF1 expression levels in Mena11a-expressing cells that
fall below the threshold needed to stimulate the pro-invasive
macrophage behavior (Fig. 2D).
MenaINV-expressing carcinoma cell streaming requires the
presence of macrophages and paracrine signaling
Because the enhanced invasion of MenaINV-expressing carcinoma
cells depends on paracrine loop signaling with macrophages, and
the paracrine loop has been shown to be required for tumor cell
migration in mammary tumors (Wyckoff et al., 2004), we
hypothesized that the paracrine loop also drives carcinoma cell
streaming in vivo. Using IVI, we observed that multiple carcinoma
cells moved in streams among host cell shadows previously
Fig. 3. Macrophages co-migrate with carcinoma cells during coordinated cell
migration as part of the migratory stream. (A)Multiphoton microscopy of
carcinoma cells (green cells outlined in white) moving coordinately in a stream with
host immune cells (black shadow outlined in orange). Images taken at 20? over 30
minutes. Arrows point to host immune cells. Arrowheads point to carcinoma cells.
Green, Cerulean MTLn3 cells expressing EGFP–MenaINV. Purple, collagen I second
harmonic. Scale bars: 25m. (B)Multiphoton microscopy of carcinoma cells (green
cells outlined in white) and Texas Red dextran-labeled macrophages (red cells
outlined in orange) moving in a stream. Green, Cerulean MTLn3 cells expressing
EGFP–MenaINV. Purple, collagen I second harmonic. Red, Texas Red-labeled
dextran. Images taken at 20? over 30 minutes. Scale bars: 25m.
(C)Immunohistochemistry of EGFP–MenaINVprimary tumor section stained for
carcinoma cells (pink) and macrophages (gray), imaged at 63?. Pink, EGFP–
MenaINVwithin MTLn3–EGFP–MenaINVcells. Gray, F4/80 within macrophages.
Green, nuclear counterstain. (D)Normalized number of cells streaming per field
quantified from IVI of primary mammary tumors derived from mammary gland
injection of MTLn3–EGFP–MenaINVtreated with the indicated reagents; 30–50
fields analyzed per condition. P values indicated above bars. Error bars indicate
Journal of Cell Science
identified as immune cells and macrophages (Wyckoff et al., 2000b;
Wyckoff et al., 2007) (Fig. 3A; supplementary material Movie 3).
Intravenous injection of Texas Red dextran during IVI indeed
confirmed the identity of the host cell shadows in tumor cell
streams as macrophages, because macrophages uniquely take up
dextran delivered intravenously into mammary tumors over the
time interval of these experiments (Wyckoff et al., 2007) (Fig. 3B;
immunohistochemistry of fixed primary tumors identified
macrophages intercalated between carcinoma cells in cell streams
To determine whether streaming required macrophages, mice
were treated with clodronate liposomes 48 hours prior to IVI to
decrease levels of functional macrophages (Hernandez et al., 2009).
A 70% decrease in the number of Texas Red dextran-labeled
macrophages was observed in animals treated with clodronate
liposomes compared with those treated with PBS liposomes
(supplementary material Fig. S5). IVI of primary tumors
demonstrated that there were 90% fewer streaming cells in mice
treated with clodronate liposomes as compared with controls,
confirming the involvement of the macrophage–tumor cell paracrine
loop in streaming (Fig. 3D). To confirm the involvement of the
paracrine loop in streaming, mice were treated with Erlotinib 2
hours prior to IVI to block the EGFR on tumor cells (Zerbe et al.,
2008) or with a CSF1R antibody 4 hours prior to IVI to block the
CSF1R on macrophages (Wyckoff et al., 2007). IVI of primary
tumors demonstrated that there were 65% fewer streaming cells in
mice treated with Erlotinib and 80% fewer streaming cells in mice
treated with -CSF1R as compared with tumors (Fig. 3D).
Movie 4). Additionally,
Expression of MenaINVin tumor cells leads to increased
transendothelial migration and intravasation
Given that our data shows increased streaming (Fig. 1B) and
invasion (Fig. 2A) in MenaINV-expressing cells we hypothesized
that expression of MenaINVmight also enhance intravasation to
increase metastasis. Following intravenous injection of Texas Red
dextran, IVI of MenaINV-expressing tumors showed that tumor cell
streaming was indeed directed toward blood vessels (Fig. 4A;
supplementary material Movie 5). We then quantified the
intravasation efficiency of tumors expressing MenaINVand
Mena11a using IVI of photoconverted carcinoma cells adjacent to
blood vessels to determine the percentage of tumor cells
intravasating over a 24-hour period (Fig. 4B) (Kedrin et al., 2008).
Quantification of the change in the photoconverted tumor area 24
hours after photoconversion showed that 95% of Mena11a-
expressing cells remained in the converted area as compared with
75% of carcinoma cells expressing MenaINV(Fig. 4Bi,ii).
Additionally, we evaluated the tumor cell blood burden to measure
intravasation in vivo (Wyckoff et al., 2000b). Mice with MenaINV-
expressing tumors had a fourfold increase in the number of
carcinoma cells in circulation compared with mice with GFP- or
Mena11a-expressing tumors (Fig. 4C). Mena11a-expressing
xenograft mice had similar numbers of circulating carcinoma cells
as compared with GFP-expressing xenograft mice (Fig. 4C).
Given that previous studies showed that interaction between
tumor cells and perivascular macrophages is required for
intravasation (Wyckoff et al., 2007), and that our studies show that
enhanced MenaINVcell streaming and invasion are paracrine-
dependent, we hypothesized that enhanced intravasation in
MenaINV-expressing cells might also be paracrine-dependent. To
examine the minimum requirements for macrophage-assisted
Mena isoforms in migration and intravasation
intravasation we used a subluminal-to-luminal transendothelial
migration (TEM) assay in which we could vary the presence of
macrophages to determine their need for carcinoma cell
intravasation (Fig. 4D). Interestingly, less than 0.5% of carcinoma
cells traversed the endothelium in the absence of macrophages,
regardless of Mena isoform expression (Fig. 4E). Addition of
macrophages did not enhance TEM for cells expressing GFP or
Mena11a (Fig. 4E). Remarkably, in the presence of macrophages,
54% of MenaINV-expressing cells traversed the endothelium, a
200-fold increase in TEM as compared with all other cell types
To test the paracrine dependence of intravasation in vivo, we
assessed the number of circulating tumor cells following functional
impairment of macrophages achieved by treatment of mice with
clodronate liposomes or CSF1R-blocking antibody, or impairment
of tumor cells by treatment with Erlotinib. Tumors formed from
injection of MenaINV-expressing cells showed a significant decrease
in circulating tumor cells following treatment with clodronate
liposomes, CSF1R blocking antibody and Erlotinib as compared
with those treated with respective controls (Fig. 4F).
Expression of MenaINV, but not Mena11a, increases
intravasation, dissemination and lung metastasis
To determine the mechanistic consequence of enhanced
transendothelial migration and intravasation by MenaINVexpression
or the suppression of invasion by Mena11a expression, we
investigated the ability of these cells to extravasate, disseminate
and metastasize to the lung. An experimental metastasis assay was
used as a measure of extravasation (Xue et al., 2006). Micro-
metastases in the lungs were counted after intravenous injection of
GFP-, Mena11a- or MenaINV-expressing cells. The metastatic
burden was similar for all cell lines (Fig. 5A).
Previous studies have shown that MTLn3 tumor cells forced to
express MenaINVshow a significant increase in the number of
metastases formed (Philippar et al., 2008). Thus, we asked whether
dissemination of single tumor cells to the lung, a step preceding
growth of macrometastases, was affected in xenograft mice derived
from injection of cells expressing MenaINVor Mena11a. Mice with
MenaINVxenografts had significantly increased carcinoma cell
dissemination to the lungs compared with animals bearing either
Mena11a- or GFP-expressing tumors (Fig. 5B). Mice with Mena11a
xenografts had half as many cells in the lungs as mice bearing
GFP-expressing tumors (Fig. 5B).
Given that MenaINV
expression increases tumor cell
dissemination and that Mena11a expression decreases tumor cell
dissemination, we hypothesized that Mena isoform expression
would similarly affect the final step in metastatic progression: the
formation of spontaneous metastasis. Spontaneous metastases to
the lungs were scored in mice with mammary tumors at either 3 or
4 weeks after mammary gland injection of GFP-, Mena11a- and
MenaINV-expressing cells. Expression of MenaINVincreased the
incidence of metastasis compared with expression of GFP and
Mena11a, whereas expression of Mena11a decreased metastases
after 3 weeks of tumor growth (Fig. 5C). However, after 4 weeks
of tumor growth, all primary tumors resulted in detectable
metastases regardless of the Mena isoform expressed (Fig. 5C). In
addition, MenaINVexpression promoted metastatic spread to the
lungs with little (at 3 weeks) or no (at 4 weeks) effect on primary
tumor growth (supplementary material Fig. S6A,B) (Philippar et
al., 2008) or cell growth in vitro (supplementary material Fig.
S6C). Hence, differences in tumor metastasis occurring in tumors
Journal of Cell Science
with different Mena isoform expression are not an indirect
consequence of tumor growth. These data indicate that the increased
incidence of spontaneous metastasis observed in MenaINV-
expressing tumors is due to metastatic events occurring prior to
Increased Mena expression is correlated with metastasis in breast
cancer patients (Di Modugno et al., 2006). In particular, during
invasion and migration of tumor cells, expression of MenaINV
increases whereas that of Mena11a decreases (Goswami et al.,
2009). In this study, we have identified invasion, migration and
intravasation as crucial steps of metastasis that are affected by
expression of MenaINV- and Mena11a-expressing tumor cells. A
key characteristic of MenaINV-expressing cells is their contribution
to cell streaming and enhanced intravasation as a result of the
dramatic increase in transendothelial migration. Another important
finding is the effect of MenaINVexpression on tumor cell sensitivity
2126 Journal of Cell Science 124 (13)
to macrophage-supplied EGF and the subsequent enhancement of
paracrine-mediated invasion. Our findings ultimately suggest that
the EGF-dependent enhancement of invasion and intravasation in
MenaINV-expressing tumor cells contributes to increased tumor
cell dissemination and spontaneous metastasis to the lungs.
Conversely, we found that Mena11a-expressing cells do not
show dramatically increased streaming and fail to co-invade with
macrophages, which indicates a reduced paracrine signaling
interaction. The decrease in CSF1 expression in Mena11a-
expressing cells contributes to impaired paracrine signaling and
leads to the observed deficits that depend on this paracrine signaling
loop in vivo, including streaming, invasion, transendothelial
migration, tumor cell dissemination and spontaneous metastasis to
During invasion, tumor cells are known to decrease their
expression of Mena11a and begin producing the MenaINVisoform
(Goswami et al., 2009). We have shown that Mena11a expression
is correlated with decreased EGF-induced in vivo invasion. We
Fig. 4. MenaINVcells promote macrophage-
dependent transendothelial migration.
(A)Multiphoton microscopy of MTLn3–EGFP–
MenaINVcells moving towards a blood vessel within the
primary mammary tumor of a mouse over 30 minutes.
Scale bars: 25m. Green, MTLn3–EGFP–MenaINV.
Red, Texas Red dextran-labeled blood vessels. White
arrow indicates direction of cell movement. Asterisk
indicates location of blood vessel. See supplementary
material Movie 5. (Bi)Quantification of percentage of
Denra2 photoconverted tumor cells remaining in the
converted area located near a vessel at 0 and 24 hours.
(Bii) Multiphoton microscopy of a primary tumor.
Images taken at 20× over 30 minutes. Red area,
Dendra2 photoconverted MenaINVtumor cells at 0 and
24 hours. Green, Dendra2–MenaINVtumor cells. White
outline, blood vessel. White bracket indicates area
evaluated at each time point. Scale bars: 50m.
(C)Average number of single cells from 1 ml of blood
from mice with GFP, MenaINVand Mena11a mammary
xenografts; n10 animals per condition. (D)Cartoon
depicting TEM assay. Pink cells, endothelial cells. Solid
gray line, Matrigel. Dotted gray line, transwell
membrane. Red cells, macrophages (BAC1.2 cells).
Green cells, carcinoma cells. (E)Quantification of TEM
of each cell type in the absence or presence of
macrophages (M); n3 experiments each done in
duplicate. (F)Average number of circulating carcinoma
cells in tumors derived from injection of MTLn3–
EGFP–MenaINVcells following the indicated treatment;
n10 experiments per condition. P values indicated
above bars. Error bars indicate s.e.m.
Journal of Cell Science
have also shown that MenaINV-expressing migratory carcinoma
cells are highly sensitive to EGF in their protrusion and chemotaxis
activities, leading to significantly enhanced in vivo invasion. These
activities can result in MenaINV-expressing cell migration towards,
and association with, perivascular macrophages, resulting in
enhanced transendothelial migration and intravasation.
In addition to decreased EGF-induced in vivo invasion of
Mena11a-expressing cells, we also found that these cells express
less CSF1 mRNA. Data from patients suggests that CSF1 and its
receptor play crucial roles during progression of breast cancer
(Kacinski et al., 1991; Scholl et al., 1994) and that CSF1 and the
CSF1R are coexpressed in >50% of breast tumors (Kacinski, 1997).
Elevated circulating CSF1 was also suggested to be an indicator of
early metastatic relapse in patients with breast cancer, independent
of breast cancer subtype (Scholl et al., 1994; Tamimi et al., 2008;
Beck et al., 2009). This suggests that lower levels of CSF1 in
Mena11a-expressing cells could lead to decreased metastatic
progression. The decreased invasion, intravasation and
dissemination of Mena11a-expressing cells are consistent with the
decrease in expression of CSF1 and the reduced sensitivity to EGF,
which would make these cells less likely to participate in a paracrine
signaling loop with macrophages.
Mena isoforms in migration and intravasation
A major finding of our study is that the expression of MenaINV
enhances a form of coordinated cell migration not previously
described, where cell migration is spatially and temporally
coordinated between carcinoma cells that are not connected by
junctions. We call this newly described form of coordinated cell
migration ‘streaming’. Streaming differs from previously described
forms of coordinated cell migration, which require the stable
retention of cell–cell junctions (Sahai, 2005), because streaming
cells need not make contact and the velocities of migration are 10–
100 times more rapid. Previous studies have shown that in vivo
MTLn3 cells express CSF1 and EGFR, but do not produce CSF1R
or EGF, whereas macrophages express CSF1R and EGF but do not
produce CSF1 or EGFR (Goswami et al., 2005). Therefore
coordinated arms of the paracrine signaling pathways are active in
both cell types during invasion in vivo (Wyckoff et al., 2004). In
our study, we demonstrate that streaming requires paracrine
chemotaxis between carcinoma cells and macrophages. The ability
of MenaINV-expressing cells to protrude and chemotax to 25- to
50-fold lower concentrations of EGF than parental tumor cells, and
to suppress cell turning in streams, undoubtedly contributes to the
extraordinary coordination and maintenance of high velocity
migration as cell streams in vivo. We propose that the increased
sensitivity of MenaINV-expressing cells to EGF in the EGF–CSF1
paracrine loop is responsible for the increase in streaming motility.
This conclusion is supported by the inhibition of streaming
following blocking of the EGFR by Erlotinib, or of CSF1R by -
CSF1R (Fig. 3D).
Invasive tumor cells from PyMT mice exhibit increased MenaINV
expression and decreased expression of Mena11a (Goswami et al.,
2009). Interestingly, recent studies using intravital imaging of
mammary tumors in Mena-deficient PyMT mice have shown
significantly decreased streaming motility of tumor cells, providing
further evidence that Mena contributes to enhanced motility
(Roussos et al., 2010). Finally, mammary tumors derived from the
human breast cancer cell line, MDA-MB-231, have tumor cells
that participate in macrophage–tumor cell paracrine-mediated
invasion (Patsialou et al., 2009), and these invasive tumor cells
have also been shown to differentially upregulate MenaINVand
downregulate Mena11a (Goswami et al., 2009). Together, these
findings suggest that paracrine-mediated carcinoma cell streaming
is a generalized phenomenon that occurs in rat, mouse and human
models of breast cancer and is a consequence of the differential
regulation of the Mena isoforms.
In our study, the suppression of invasion and streaming by the
inhibition of paracrine signaling between macrophages and tumor
cells in vivo, and by decreasing macrophage function in vivo,
demonstrates the crucial role of macrophages during coordinated
migration of MenaINV-expressing cells (Wyckoff et al., 2007;
Hernandez et al., 2009). We also demonstrate that macrophages are
essential for transendothelial migration of MenaINV-expressing
tumor cells. Our results are consistent with previous work showing
that paracrine signaling between tumor cells and macrophages, and
the presence of perivascular macrophages in the primary tumor,
are required for invasion and intravasation, respectively (Wyckoff
et al., 2004; Wyckoff et al., 2007). In particular, our results support
previous work suggesting that MenaINV- but not Mena11a-
expressing tumor cells specifically contribute to intravasation of
breast cancer cells in humans by helping to assemble the
macrophage-dependent intravasation compartment called TMEM
(Robinson et al., 2009; Roussos et al., 2011).
Fig. 5. MenaINVenhances dissemination of tumor cells and spontaneous
metastasis to the lungs. (A)Experimental lung metastasis quantified after
intravenous injection of GFP-, Mena11a- or MenaINV-expressing cells showing
no statistically significant difference; n10 animals per cell type. (B)Average
number of single tumor cells disseminated into the lungs of mice with GFP,
Mena11a or MenaINVmammary xenografts; n10 animals per condition.
(C)Number of animals with spontaneous lung metastases in mice with GFP,
Mena11a and MenaINVmammary xenografts following primary tumor growth.
P values indicated above bars. Error bars indicate s.e.m.
Journal of Cell Science
2128 Journal of Cell Science 124 (13)
In vivo, we have shown that MenaINV-expressing cells invade
towards very low concentrations of EGF in macrophage-dependent
paracrine chemotaxis. In vitro, low concentrations of EGF such as
that found in serum, lead to macrophage-independent 3D invasion
of MenaINV-expressing cells (Philippar et al., 2008), whereas
completion of transendothelial migration requires EGF supplied
by macrophages (Wyckoff et al., 2004). The effects of MenaINV
expression on EGF-dependent processes lead to increased invasion,
intravasation, dissemination and metastasis to the lungs. These
data suggest that drugs directed specifically to the inhibition of
MenaINV-dependent increased EGF sensitivity will disrupt the
paracrine interactions with macrophages required for metastasis,
and result in the inhibition of metastasis in mammary tumors.
In this regard, it will be important to understand how the Mena
isoforms differ functionally. The INV exon is inserted just after the
EVH1 domain, which is primarily responsible for the subcellular
localization of Ena/VASP proteins and interactions with several
signaling proteins such as Lamellipodin (Gertler et al., 1996;
Urbanelli et al., 2006; Pula and Krause, 2008). It is therefore
possible that the INV exon might influence the function of MenaINV
by regulating its EVH1-mediated interactions (Niebuhr et al., 1997;
Boeda et al., 2007). The 11a exon is inserted within the EVH2
domain between the F-actin binding motif and the coiled-coil
tetramerization site. F-actin binding is crucial for almost all known
Ena/VASP functions, including localization to the tips of
lamellipods and the ability to drive filopod and lamellipod
formation and extension (Gertler et al., 1996; Loureiro et al., 2002;
Applewhite et al., 2007). In vitro, F-actin binding is required for
the anti-capping activity of Ena/VASP and is disrupted by
phosphorylation at nearby sites (Barzik et al., 2005), as is F-actin
bundling. Because 11a is inserted in the analogous region of Mena,
it will be interesting to determine whether the barbed end capture
activity is affected. Because the 11a insertion is phosphorylated
(Di Modugno et al., 2006), it is possible that inclusion of the 11a
exon provides a regulatory mechanism for Mena11a.
Future studies will be needed to investigate the molecular and
biochemical mechanisms of action of the MenaINVand Mena11a
isoforms and their potential utility as a prognostic marker for
patient outcome, and as a therapeutic target for breast cancer
Materials and Methods
We used MTLn3 cells derived from metastatic lung lesions from rat mammary
adenocarcinoma derived following
dimethylbenzanthracene (tumor line 13762) (Neri et al., 1982). The in vivo
biologic characteristics and metastatic potential of these cells have been determined
in previous studies and show that MTLn3 cells have a high metastatic potential (Neri
et al., 1982; Welch et al., 1983). Western blots, immunofluorescence and fluorescence-
activated cell sorting (FACS) confirm that MTLn3 cells express ErbB2, ErbB3
(Levea et al., 2000; Xue et al., 2006; Kedrin et al., 2009) and ErbB4 (personal
communication Sumanta Goswami, Yeshiva University, and Michele Balsamo, MIT,
Cambridge, MA). Previous studies have also confirmed that EGFRs in MTLn3 cells
are fully active and homogenously distributed on the plasma membrane (Lichtner et
al., 1992; Bailly et al., 2000). Dominant coexpression of vimentin and CK14 in
MTLn3 cells suggests that these cells represent a myoepithelial or basal cell that has
partially dedifferentiated (Lichtner et al., 1991). Additionally, MTLn3 cells have
been shown to have increased expression of EGFR as compared with the non-
metastatic MTC clone derived from the same mouse model (Lichtner et al., 1995;
Levea et al., 2000). Taken together, these data indicate that MTLn3 cells have a
phenotype similar to basal-like tumors. MTLn3 cells were used in these experiments
because they are known to metastasize to the lung when injected into the mammary
gland of SCID mice and thus are suitable for metastatic studies. Additionally, these
cells have been used to study metastasis by several laboratories, and Mena isoforms
in particular (Wyckoff et al., 2000b; Wyckoff et al., 2000a; Sahai, 2005; Philippar et
al., 2008; Le Devedec et al., 2010).
dietary administration of 7,12-
Molecular cloning, infection, FACS and cell culture
EGFP–Mena splice isoforms were subcloned into the retroviral vector packaging
Murine stem cell virus–EGFP using standard techniques. MTLn3 cells were used for
all of the experiments described. MTLn3–Cerulean EGFP–Mena splice isoforms
were created using a lentiviral system pCCLsin.PPT.hPGK.Cerulean.pre (courtesy
of Sanjeev Gupta, Albert Einstein College of Medicine). MTLn3-Dendra2 cells were
created using a Dendra2 cloning vector C1 with G418 selection marker (courtesy of
Vladislav Verkhusha, Albert Einstein College of Medicine). Transfection of Dendra2
into MTLn3–EGFP–MenaINVand MTLn3–EGFP–Mena11a cells lines was done
using Lipofectamine 2000 (Invitrogen). Retroviral packaging was performed as
previously described (Bear et al., 2000). MTLn3–EGFP–MenaINVand MTLn3–
EGFP–Mena11a cell lines were FACS sorted to a level of fourfold overexpression
of each fusion protein. Sorting of all other cells was done using FACS 72 hours after
transfection. The 10% brightest population of infected cell lines were kept for
culturing, and selection was maintained using 500 g/ml G418 geneticin (Invitrogen)
when necessary. MTLn3 cells were cultured in -modified minimum essential
medium (-MEM) supplemented with 5% fetal bovine serum (FBS) and 0.5%
Animal model and assays for metastatic progression
In vivo studies were performed in orthotopic tumors derived from injection of
MTLn3 rat adenocarcinoma cells into SCID mice. MTLn3 cells were engineered to
express either EGFP (for controls) or Mena isoform EGFP-fusion proteins: EGFP–
MenaINV(MenaINV) and EGFP–Mena11a (Mena11a) at roughly four times the
normal level, consistent with the level of spontaneous Mena upregulation in invasive
tumor cells in vivo (Philippar et al., 2008; Goswami et al., 2009). SCID mice with
tumors derived from injection of these cells into the mammary gland are referred to
as GFP, MenaINVor Mena11a xenografts. Tumor cell blood burden was evaluated
following 4 weeks of tumor growth as previously described (Wyckoff et al., 2000b).
Briefly, blood was drawn from the right ventricle of anesthetized mice and cells were
plated in -MEM media. Following 7 days of cell culture, tumor cells were counted.
All cells counted were GFP-positive, confirming their identity as tumor cells. To
functionally impair macrophages, mice were treated with 100 l of PBS (control) or
clodronate (Cl2MDP)-containing liposomes per 10 g of weight via tail vein injection
24 hours prior to collection of blood (Hernandez et al., 2009). Liposomes were
prepared as previously described using a clodronate concentration of 2.5 g/10 ml of
PBS (van Rooijen and van Kesteren-Hendrikx, 2003). Clodronate was a gift of
Roche Diagnostics (Mannheim, Germany). Phosphatidylcholine (Lipoid E PC) was
obtained from Lipoid (Ludwigshafen, Germany). Cholesterol was purchased from
Sigma. Mice were treated with CSF1R blocking antibody or IgG antibody 4 hours
prior to collection of blood to block signaling between tumor cells and macrophages
(Wyckoff et al., 2004). Single tumor cell dissemination to the lung was quantified
ex vivo using epifluorescence in ten random high power fields per set of mouse
lungs at 3 weeks after mammary gland injection of each cell line. Spontaneous lung
metastases (>2 mm) were evaluated ex vivo using epifluorescence at 3 and 4 weeks
after mammary gland injection of MenaINV- or Mena11a-expressing cells as
previously described (Philippar et al., 2008). Experimental metastases were evaluated
ex vivo 2 weeks after tail vein injection of 5?105cells of each cell type as previously
described (Wang et al., 2006). Lungs were examined using a 60? 1.2 NA water
immersion correction lens on an inverted Olympus IX70 (Wyckoff et al., 2000b).
For each experiment described above, 10–15 animals were used per Mena isoform
cell type. All experiments involving animals were approved by the Einstein Institute
for Animal Studies.
In vivo invasion assay and in vitro 3D invasion assay
The in vivo invasion assay was performed in 5–10 mice per condition as previously
described (Wyckoff et al., 2000a). We have previously measured the ranges of EGF
and CSF1 concentrations required to initiate chemotaxis and migration in vivo
(Wyckoff et al., 2004; Patsialou et al., 2009; Raja et al., 2010). The concentrations
shown to be effective and used in our study vary from 50% to 10% of the Kdof these
ligands for their respective receptors. In addition, these are the concentrations
believed to be present in vivo (Byyny et al., 1974; Bartocci et al., 1986). The
paracrine loop was inhibited using 10 g of affinity-purified -mouse CSF1R-
blocking antibody (-CSF1R; courtesy of Richard Stanley, Yeshiva University,
Bronx, NY) or 6.25 nM Erlotinib (gift from OSI Pharmaceuticals, Melvile, NY),
empirically determined by OSI Pharmaceuticals, dissolved in DMSO in needles
containing 25 nM and 1nM EGF for GFP- and MenaINV-expressing cells, respectively
(Wyckoff et al., 2004); -rat IgG or DMSO were used as controls, respectively. Cells
were imaged on an Olympus IX70 inverted microscope with a 10? NA 0.30
objective. For in vivo invasion experiments performed with EGFR (Erlotinib) and
CSF1R inhibitors, we used 1nM EGF in the needles inserted into MenaINV-expressing
tumors and 25nM EGF in the needles inserted into GFP-expressing tumors to allow
maximal tumor cell collection (350–600 cells collected per needle) (Philippar et al.,
2008). In vitro 3D invasion assays were performed as previously described (Goswami
et al., 2005).
Intravital multiphoton imaging was performed as described previously (Wang et al.,
2002; Wyckoff et al., 2010) using a 20? 1.95 NA water immersion objective with
Journal of Cell Science
correction lens. Time-lapse movies were analyzed for frequency of motility and
tracking, and for measuring and quantifying of cell characteristics in 3D and through
time using NIH ImageJ (Sahai et al., 2005) and custom software described elsewhere
(Wyckoff et al., 2010). A cell movement event was defined as a translocation of >1
cell diameter (25m) observed within a visual field that is defined in three dimensions
as 100 m by 512?512 pixels per minute. Streaming cells were quantified as the
number of individual carcinoma cells in a field whose vector paths point in the same
direction (Fig. 1A). Random cells movements were quantified as the number of
individual carcinoma cells in a field whose vector paths point in different directions
(Fig. 1A). To confirm the elimination of macrophages in clodronate-treated mice,
multiphoton microscopy of spleens removed from both clodronate- and PBS
liposome-treated animals was performed as previously described (Hernandez et al.,
2009). Mice were given intraperitoneal injection of 10 mg/kg body weight of vehicle
(6% Captisol) or Erlotinib in 6% Captisol, 2 hours prior to IVI, or 2.5 g of CSF1R-
blocking anitbody or IgG in PBS 4 hours prior to IVI. Tail vein injection of 200 l
70 kDa Texas Red dextran was used to label blood vessels, which were immediately
visualized following injection. After 2 hours, macrophages were also labeled with
dextran as previously described (Wyckoff et al., 2007).
Mammary imaging windows were placed over the mammary gland of mice 3
weeks after injection of carcinoma cells (Kedrin et al., 2008). Photoconversion of
the Dendra2 variant from green to red was done using a 405 nm UV laser on a Leica
TCS SP2 AOBS confocal microscope (Mannheim, Germany) equipped with a 20?
glycerol objective (Kedrin et al., 2008). Z-stacks (~100 m deep) of the same fields
were imaged at 0 and 24 hours after photoconversion in imaging sessions using a
multiphoton microscope equipped with a MaiTai 15 W laser tuned to 1045 nm for
red Dendra2 and a Tsunami 15 W laser tuned to 880 nm for green Dendra2 (Kedrin
et al., 2008). Quantification of intravasation was done as previously described
(Kedrin et al., 2008).
Real time PCR
RNA was extracted from all cell lines at steady state, and quantitative real time PCR
was performed as previously described (Goswami et al., 2009). Primers designed
against Rat EGFR and CSF1 were used to determine levels of expression of each.
Primer sequences used for EGFR: rEGFR forward 5?-TCGTTGCCGACGCAGT-
CACC-3?, rEGFR reverse 5?-TCCCTGAGGGTCGCATCCCG-3?. Primer sequences
used for CSF1: rCSF-1 forward 5?-GCTCGAGGGCAAGAAAAGTA-3?, rCSF-1
Transendothelial migration assay
To prepare the endothelial monolayer, the underside of each transwell was coated
with 50 l of 2.5 g/ml Matrigel (Invitrogen). Some 200,000 rat pulmonary
microvascular endothelial cells (RLE) were plated in 50 l of DMEM + 10% FBS
(medium 1) and incubated at 37°C for 4 hours. Transwells were flipped onto a 24-
well plate containing 1 or 200 l of medium 1 in the lower or upper chambers,
respectively. BAC1.2F5 macrophages and tumor cells were labeled with cell-tracker
dyes. Then, 15,000 macrophages and 37,500 tumor cells were added to the upper
chamber in 200 l of -MEM + 0.5% FBS (medium 2), and 200 l of -MEM +
10% FBS + 3000 units CSF1 were added to the lower chamber. Following 18 hours
of transmigration, medium was removed from the top of the transwell and migrated
cells were scraped from the bottom of the plate. Cells were fixed in 0.1%
formaldehyde and analyzed using a Guava flow cytometer. Monolayers were tested
for permeability both before and after cell migration via quantification of fluorescence
(Molecular Devices SpectraMax M5 plate reader) of 70 kDa rhodamine-dextran in
the media from both upper and lower transwell chambers added 24 hours prior.
Primary tumors were fixed in 10% buffered formalin, and paraffin embedded.
Sections of 10 m were cut and placed on slides for further staining.
Immunohistochemistry was carried out using -GFP at 1:200 to identify tumor cells
containing GFP-fusion proteins, and rat -F4/80 (courtesy of Jeffrey Pollard, Albert
Einstein College of Medicine) was used at 1:25 to identify macrophages. All staining
was done using standard protocols, and eosin was used as a nuclear counterstain.
Slides were analyzed and imaged using Zeiss AxioObserver.Z1 5? DIC1, EC Plan-
Neofluar 10?/0.3 Ph1, EC Plan-Neofluar 20?/0.5 Ph2 M27, EC Plan-Neofluar
40?/0.75 Ph2 M27, EC Plan-neofluar 63?/1.4 Oil and an AxioCamHR3.
For all experiments, statistical significances were determined using unpaired, two-
tailed Student’s t-tests assuming equal variances and an alpha level of 0.05 unless
otherwise specified. Differences were considered significant for P<0.05. For
assessment of lung metastasis and circulating tumor cells, the non-parametric Mann
Whitney Wilcoxon rank sum test was used.
We would like to thank Diane Cox, Antonia Patsialou, and Daqian
Sun for stimulating discussion and helpful suggestions. We would also
like to thank Richard Stanley (Albert Einstein College of Medicine,
Yeshiva University, Bronx, NY) for his generous contribution of
CSF1R blocking antibody as well as OSI Pharmaceuticals (Melville,
Mena isoforms in migration and intravasation
NY) for their generous donation of Erlotinib. Many thanks to David
Entenberg and Jenny Tadros for their technical support, Einstein
histopathology and analytical imaging facilities and Koch Institute
Microscopy core facility for their services. Grant support was provided
by NIH-CA100324 (A.R.B., J.B.W., Y.W. S.G., J.E.S.), NIH-CA126511
(B.G.), NIH-CA150344 (E.T.R., J.S.C.), NIH-CA77522 (J.E.S.),
Ludwig Fund postdoctoral fellowship (M.B.), DoD CDMRP BCRP
Fellowship (BC087781) (S.K.A.), NIH-GM58801 and funds from the
Ludwig Center at MIT (F.B.G.), ICBP grant U54 CA112967 (D.A.L.,
F.B.G.) and the Charles H. Revson Fellowship (B.G.). None of the
authors have any conflicts of interest. Data in this paper are from a
thesis to be submitted in partial fulfillment of the requirements for the
Degree of Doctor of Philosophy in the Graduate Division of Medical
Sciences, Albert Einstein College of Medicine, Yeshiva University.
Deposited in PMC for release after 12 months.
Supplementary material available online at
Andresen, V., Alexander, S., Heupel, W. M., Hirschberg, M., Hoffman, R. M. and
Friedl, P. (2009). Infrared multiphoton microscopy: subcellular-resolved deep tissue
imaging. Curr. Opin. Biotechnol. 20, 54-62.
Applewhite, D. A., Barzik, M., Kojima, S., Svitkina, T. M., Gertler, F. B. and Borisy,
G. G. (2007). Ena/VASP proteins have an anti-capping independent function in filopodia
formation. Mol. Biol. Cell 18, 2579-2591.
Bailly, M., Wyckoff, J., Bouzahzah, B., Hammerman, R., Sylvestre, V., Cammer, M.,
Pestell, R. and Segall, J. E. (2000). Epidermal growth factor receptor distribution
during chemotactic responses. Mol. Biol. Cell 11, 3873-3883.
Bartocci, A., Pollard, J. W. and Stanley, E. R. (1986). Regulation of colony-stimulating
factor 1 during pregnancy. J. Exp. Med. 164, 956-961.
Barzik, M., Kotova, T. I., Higgs, H. N., Hazelwood, L., Hanein, D., Gertler, F. B. and
Schafer, D. A. (2005). Ena/VASP proteins enhance actin polymerization in the presence
of barbed end capping proteins. J. Biol. Chem. 280, 28653-28662.
Bear, J. E. and Gertler, F. B. (2009). Ena/VASP: towards resolving a pointed controversy
at the barbed end. J. Cell Sci. 122, 1947-1953.
Bear, J. E., Loureiro, J. J., Libova, I., Fassler, R., Wehland, J. and Gertler, F. B.
(2000). Negative regulation of fibroblast motility by Ena/VASP proteins. Cell 101, 717-
Bear, J. E., Svitkina, T. M., Krause, M., Schafer, D. A., Loureiro, J. J., Strasser, G.
A., Maly, I. V., Chaga, O. Y., Cooper, J. A., Borisy, G. G. et al. (2002). Antagonism
between Ena/VASP proteins and actin filament capping regulates fibroblast motility.
Cell 109, 509-521.
Beck, A. H., Espinosa, I., Edris, B., Li, R., Montgomery, K., Zhu, S., Varma, S.,
Marinelli, R. J., van de Rijn, M. and West, R. B. (2009). The macrophage colony-
stimulating factor 1 response signature in breast carcinoma. Clin. Cancer Res. 15, 778-
Boeda, B., Briggs, D. C., Higgins, T., Garvalov, B. K., Fadden, A. J., McDonald, N.
Q. and Way, M. (2007). Tes, a specific Mena interacting partner, breaks the rules for
EVH1 binding. Mol. Cell 28, 1071-1082.
Byyny, R. L., Orth, D. N., Cohen, S. and Doyne, E. S. (1974). Epidermal growth factor:
effects of androgens and adrenergic agents. Endocrinology 95, 776-782.
Condeelis, J. and Pollard, J. W. (2006). Macrophages: obligate partners for tumor cell
migration, invasion, and metastasis. Cell 124, 263-266.
Condeelis, J. and Segall, J. E. (2003). Intravital imaging of cell movement in tumours.
Nat. Rev. Cancer 3, 921-930.
Di Modugno, F., Mottolese, M., Di Benedetto, A., Conidi, A., Novelli, F., Perracchio,
L., Venturo, I., Botti, C., Jager, E., Santoni, A. et al. (2006). The cytoskeleton
regulatory protein hMena (ENAH) is overexpressed in human benign breast lesions
with high risk of transformation and human epidermal growth factor receptor-2-
positive/hormonal receptor-negative tumors. Clin. Cancer Res. 12, 1470-1478.
Di Modugno, F., DeMonte, L., Balsamo, M., Bronzi, G., Nicotra, M. R., Alessio, M.,
Jager, E., Condeelis, J. S., Santoni, A., Natali, P. G. et al. (2007). Molecular cloning
of hMena (ENAH) and its splice variant hMena+11a: epidermal growth factor increases
their expression and stimulates hMena+11a phosphorylation in breast cancer cell lines.
Cancer Res. 67, 2657-2665.
Egeblad, M., Ewald, A. J., Askautrud, H. A., Truitt, M. L., Welm, B. E., Bainbridge,
E., Peeters, G., Krummel, M. F. and Werb, Z. (2008). Visualizing stromal cell
dynamics in different tumor microenvironments by spinning disk confocal microscopy.
Dis. Model. Mech. 1, 155-167; discussion 165.
Ferron, F., Rebowski, G., Lee, S. H. and Dominguez, R. (2007). Structural basis for the
recruitment of profilin-actin complexes during filament elongation by Ena/VASP. EMBO
J. 26, 4597-4606.
Friedl, P. and Wolf, K. (2010). Plasticity of cell migration: a multiscale tuning model. J.
Cell Biol. 188, 11-19.
Gaggioli, C., Hooper, S., Hidalgo-Carcedo, C., Grosse, R., Marshall, J. F., Harrington,
K. and Sahai, E. (2007). Fibroblast-led collective invasion of carcinoma cells with
differing roles for RhoGTPases in leading and following cells. Nat. Cell Biol. 9, 1392-
Journal of Cell Science
Gertler, F. B., Niebuhr, K., Reinhard, M., Wehland, J. and Soriano, P. (1996). Mena,
a relative of VASP and Drosophila Enabled, is implicated in the control of microfilament
dynamics. Cell 87, 227-239.
Giampieri, S., Manning, C., Hooper, S., Jones, L., Hill, C. S. and Sahai, E. (2009).
Localized and reversible TGFbeta signalling switches breast cancer cells from cohesive
to single cell motility. Nat. Cell Biol. 11, 1287-1296.
Goswami, S., Wang, W., Wyckoff, J. B. and Condeelis, J. S. (2004). Breast cancer cells
isolated by chemotaxis from primary tumors show increased survival and resistance to
chemotherapy. Cancer Res. 64, 7664-7667.
Goswami, S., Sahai, E., Wyckoff, J. B., Cammer, M., Cox, D., Pixley, F. J., Stanley, E.
R., Segall, J. E. and Condeelis, J. S. (2005). Macrophages promote the invasion of
breast carcinoma cells via a colony-stimulating factor-1/epidermal growth factor
paracrine loop. Cancer Res. 65, 5278-5283.
Goswami, S., Philippar, U., Sun, D., Patsialou, A., Avraham, J., Wang, W., Di Modugno,
F., Nistico, P., Gertler, F. B. and Condeelis, J. S. (2009). Identification of invasion
specific splice variants of the cytoskeletal protein Mena present in mammary tumor
cells during invasion in vivo. Clin. Exp. Metastasis 26, 153-159.
Gurzu, S., Jung, I., Prantner, I., Ember, I., Pavai, Z. and Mezei, T. (2008). The
expression of cytoskeleton regulatory protein Mena in colorectal lesions. Rom. J.
Morphol. Embryol. 49, 345-349.
Gurzu, S., Jung, I., Prantner, I., Chira, L. and Ember, I. (2009). The
immunohistochemical aspects of protein Mena in cervical lesions. Rom. J. Morphol.
Embryol. 50, 213-216.
Hansen, S. D. and Mullins, R. D. (2010). VASP is a processive actin polymerase that
requires monomeric actin for barbed end association. J. Cell Biol. 191, 571-584.
Hernandez, L., Smirnova, T., Kedrin, D., Wyckoff, J., Zhu, L., Stanley, E. R., Cox,
D., Muller, W. J., Pollard, J. W., Van Rooijen, N. et al. (2009). The EGF/CSF-1
paracrine invasion loop can be triggered by heregulin beta1 and CXCL12. Cancer Res.
Ilina, O. and Friedl, P. (2009). Mechanisms of collective cell migration at a glance. J.
Cell Sci. 122, 3203-3208.
Kacinski, B. M. (1997). CSF-1 and its receptor in breast carcinomas and neoplasms of the
female reproductive tract. Mol. Reprod. Dev. 46, 71-74.
Kacinski, B. M., Scata, K. A., Carter, D., Yee, L. D., Sapi, E., King, B. L., Chambers,
S. K., Jones, M. A., Pirro, M. H., Stanley, E. R. et al. (1991). FMS (CSF-1 receptor)
and CSF-1 transcripts and protein are expressed by human breast carcinomas in vivo
and in vitro. Oncogene 6, 941-952.
Kedrin, D., van Rheenen, J., Hernandez, L., Condeelis, J. and Segall, J. E. (2007).
Cell motility and cytoskeletal regulation in invasion and metastasis. J. Mammary Gland
Biol. Neoplasia 12, 143-152.
Kedrin, D., Gligorijevic, B., Wyckoff, J., Verkhusha, V. V., Condeelis, J., Segall, J. E.
and van Rheenen, J. (2008). Intravital imaging of metastatic behavior through a
mammary imaging window. Nat. Methods 5, 1019-1021.
Kedrin, D., Wyckoff, J., Boimel, P. J., Coniglio, S. J., Hynes, N. E., Arteaga, C. L. and
Segall, J. E. (2009). ERBB1 and ERBB2 have distinct functions in tumor cell invasion
and intravasation. Clin. Cancer Res. 15, 3733-3739.
Le Devedec, S. E., van Roosmalen, W., Maria, N., Grimbergen, M., Pont, C., Lalai,
R. and van de Water, B. (2009). An improved model to study tumor cell autonomous
metastasis programs using MTLn3 cells and the Rag2(–/–) gammac (–/–) mouse. Clin.
Exp. Metastasis 26, 673-684.
Le Devedec, S. E., Lalai, R., Pont, C., de Bont, H. and van de Water, B. (2010). Two-
photon intravital multicolor imaging combined with inducible gene expression to
distinguish metastatic behavior of breast cancer cells in vivo. Mol. Imaging Biol. 13,
Levea, C. M., McGary, C. T., Symons, J. R. and Mooney, R. A. (2000). PTP LAR
expression compared to prognostic indices in metastatic and non-metastatic breast
cancer. Breast Cancer Res. Treat. 64, 221-228.
Lichtner, R. B., Julian, J. A., Glasser, S. R. and Nicolson, G. L. (1989). Characterization
of cytokeratins expressed in metastatic rat mammary adenocarcinoma cells. Cancer
Res. 49, 104-111.
Lichtner, R. B., Julian, J. A., North, S. M., Glasser, S. R. and Nicolson, G. L. (1991).
Coexpression of cytokeratins characteristic for myoepithelial and luminal cell lineages
in rat 13762NF mammary adenocarcinoma tumors and their spontaneous metastases.
Cancer Res. 51, 5943-5950.
Lichtner, R. B., Wiedemuth, M., Kittmann, A., Ullrich, A., Schirrmacher, V. and
Khazaie, K. (1992). Ligand-induced activation of epidermal growth factor receptor in
intact rat mammary adenocarcinoma cells without detectable receptor phosphorylation.
J. Biol. Chem. 267, 11872-11880.
Lichtner, R. B., Kaufmann, A. M., Kittmann, A., Rohde-Schulz, B., Walter, J.,
Williams, L., Ullrich, A., Schirrmacher, V. and Khazaie, K. (1995). Ligand mediated
activation of ectopic EGF receptor promotes matrix protein adhesion and lung
colonization of rat mammary adenocarcinoma cells. Oncogene 10, 1823-1832.
Loureiro, J. J., Rubinson, D. A., Bear, J. E., Baltus, G. A., Kwiatkowski, A. V. and
Gertler, F. B. (2002). Critical roles of phosphorylation and actin binding motifs, but
not the central proline-rich region, for Ena/vasodilator-stimulated phosphoprotein
(VASP) function during cell migration. Mol. Biol. Cell 13, 2533-2546.
Neri, A., Welch, D., Kawaguchi, T. and Nicolson, G. L. (1982). Development and
biologic properties of malignant cell sublines and clones of a spontaneously metastasizing
rat mammary adenocarcinoma. J. Natl. Cancer Inst. 68, 507-517.
Niebuhr, K., Ebel, F., Frank, R., Reinhard, M., Domann, E., Carl, U. D., Walter, U.,
Gertler, F. B., Wehland, J. and Chakraborty, T. (1997). A novel proline-rich motif
present in ActA of Listeria monocytogenes and cytoskeletal proteins is the ligand for
the EVH1 domain, a protein module present in the Ena/VASP family. EMBO J. 16,
2130 Journal of Cell Science 124 (13)
Pasic, L., Kotova, T. and Schafer, D. A. (2008). Ena/VASP proteins capture actin filament
barbed ends. J. Biol. Chem. 283, 9814-9819.
Patsialou, A., Wyckoff, J., Wang, Y., Goswami, S., Stanley, E. R. and Condeelis, J. S.
(2009). Invasion of human breast cancer cells in vivo requires both paracrine and
autocrine loops involving the Colony-Stimulating Factor-1 receptor. Cancer Res. 69,
Perentes, J. Y., McKee, T. D., Ley, C. D., Mathiew, H., Dawson, M., Padera, T. P.,
Munn, L. L., Jain, R. K. and Boucher, Y. (2009). In vivo imaging of extracellular
matrix remodeling by tumor-associated fibroblasts. Nat. Methods 6, 143-145.
Philippar, U., Roussos, E. T., Oser, M., Yamaguchi, H., Kim, H. D., Giampieri, S.,
Wang, Y., Goswami, S., Wyckoff, J. B., Lauffenburger, D. A. et al. (2008). A Mena
invasion isoform potentiates EGF-induced carcinoma cell invasion and metastasis. Dev.
Cell 15, 813-828.
Pino, M. S., Balsamo, M., Di Modugno, F., Mottolese, M., Alessio, M., Melucci, E.,
Milella, M., McConkey, D. J., Philippar, U., Gertler, F. B. et al. (2008). Human
Mena+11a isoform serves as a marker of epithelial phenotype and sensitivity to
epidermal growth factor receptor inhibition in human pancreatic cancer cell lines. Clin.
Cancer Res. 14, 4943-4950.
Pula, G. and Krause, M. (2008). Role of Ena/VASP proteins in homeostasis and disease.
Handb. Exp. Pharmacol. 186, 39-65.
Raja, W. K., Gligorijevic, B., Wyckoff, J., Condeelis, J. S. and Castracane, J. (2010).
A new chemotaxis device for cell migration studies. Integr. Biol. (Camb.) 2, 696-706.
Robinson, B. D., Sica, G. L., Liu, Y. F., Rohan, T. E., Gertler, F. B., Condeelis, J. S.
and Jones, J. G. (2009). Tumor microenvironment of metastasis in human breast
carcinoma: a potential prognostic marker linked to hematogenous dissemination. Clin.
Cancer Res. 15, 2433-2441.
Roussos, E. T., Goswami, S., Balsamo, M., Wang, Y., Stobezki, R., Adler, E., Robinson,
B. D., Jones, J. G., Gertler, F. B., Condeelis J. S. et al. (2011). Mena invasive
(MenaINV) and Mena11a isoforms play distinct roles in breast cancer cell cohesion
and association with TMEM. Clin. Exp. Metastasis [Epub ahead of print] doi:
Roussos, E. T., Wang, Y., Wyckoff, J. B., Sellers, R. S., Wang, W., Li, J., Pollard, J.
W., Gertler, F. B. and Condeelis, J. S. (2010). Mena deficiency delays tumor
progression and decreases metastasis in polyoma middle-T transgenic mouse mammary
tumors. Breast Cancer Res. 12, R101.
Sahai, E. (2005). Mechanisms of cancer cell invasion. Curr. Opin. Genet. Dev. 15, 87-96.
Sahai, E., Wyckoff, J., Philippar, U., Segall, J. E., Gertler, F. and Condeelis, J. (2005).
Simultaneous imaging of GFP, CFP and collagen in tumors in vivo using multiphoton
microscopy. BMC Biotechnol. 5, 14.
Scholl, S. M., Pallud, C., Beuvon, F., Hacene, K., Stanley, E. R., Rohrschneider, L.,
Tang, R., Pouillart, P. and Lidereau, R. (1994). Anti-colony-stimulating factor-1
antibody staining in primary breast adenocarcinomas correlates with marked
inflammatory cell infiltrates and prognosis. J. Natl. Cancer Inst. 86, 120-126.
Segall, J. E., Tyerech, S., Boselli, L., Masseling, S., Helft, J., Chan, A., Jones, J. and
Condeelis, J. (1996). EGF stimulates lamellipod extension in metastatic mammary
adenocarcinoma cells by an actin-dependent mechanism. Clin. Exp. Metastasis 14, 61-
Tamimi, R. M., Brugge, J. S., Freedman, M. L., Miron, A., Iglehart, J. D., Colditz, G.
A. and Hankinson, S. E. (2008). Circulating colony stimulating factor-1 and breast
cancer risk. Cancer Res. 68, 18-21.
Urbanelli, L., Massini, C., Emiliani, C., Orlacchio, A. and Bernardi, G. (2006).
Characterization of human Enah gene. Biochim. Biophys. Acta 1759, 99-107.
van Rooijen, N. and van Kesteren-Hendrikx, E. (2003). “In vivo” depletion of
macrophages by liposome-mediated “suicide”. Methods Enzymol. 373, 3-16.
Wang, W., Wyckoff, J. B., Frohlich, V. C., Oleynikov, Y., Huttelmaier, S., Zavadil, J.,
Cermak, L., Bottinger, E. P., Singer, R. H., White, J. G. et al. (2002). Single cell
behavior in metastatic primary mammary tumors correlated with gene expression
patterns revealed by molecular profiling. Cancer Res. 62, 6278-6288.
Wang, W., Goswami, S., Lapidus, K., Wells, A. L., Wyckoff, J. B., Sahai, E., Singer,
R. H., Segall, J. E. and Condeelis, J. S. (2004). Identification and testing of a gene
expression signature of invasive carcinoma cells within primary mammary tumors.
Cancer Res. 64, 8585-8594.
Wang, W., Mouneimne, G., Sidani, M., Wyckoff, J., Chen, X., Makris, A., Goswami,
S., Bresnick, A. R. and Condeelis, J. S. (2006). The activity status of cofilin is directly
related to invasion, intravasation, and metastasis of mammary tumors. J. Cell Biol. 173,
Wang, W., Wyckoff, J. B., Goswami, S., Wang, Y., Sidani, M., Segall, J. E. and
Condeelis, J. S. (2007). Coordinated regulation of pathways for enhanced cell motility
and chemotaxis is conserved in rat and mouse mammary tumors. Cancer Res. 67, 3505-
Welch, D. R., Neri, A. and Nicolson, G. L. (1983). Comparison of ‘spontaneous’ and
‘experimental’ metastasis using rat 13762 mammary adenocarcinoma metastatic cell
clones. Invasion Metastasis 3, 65-80.
Wolf, K., Mazo, I., Leung, H., Engelke, K., von Andrian, U. H., Deryugina, E. I.,
Strongin, A. Y., Brocker, E. B. and Friedl, P. (2003). Compensation mechanism in
tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular
proteolysis. J. Cell Biol. 160, 267-277.
Wyckoff, J., Wang, W., Lin, E. Y., Wang, Y., Pixley, F., Stanley, E. R., Graf, T.,
Pollard, J. W., Segall, J. and Condeelis, J. (2004). A paracrine loop between tumor
cells and macrophages is required for tumor cell migration in mammary tumors. Cancer
Res. 64, 7022-7029.
Wyckoff, J., Gligoijevic, B., Entenberg, D., Segall, J. and Condeelis J. (2010). High-
resolution multiphoton imaging of tumors in vivo. In Live Cell Imaging: A Laboratory
Journal of Cell Science
2131 Download full-text
Mena isoforms in migration and intravasation
Manual, 2nd edn (ed. R. D. Goldman, J. R. Swedlow and D. L. Spector), pp. 441-462.
Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
Wyckoff, J. B., Segall, J. E. and Condeelis, J. S. (2000a). The collection of the motile
population of cells from a living tumor. Cancer Res. 60, 5401-5404.
Wyckoff, J. B., Jones, J. G., Condeelis, J. S. and Segall, J. E. (2000b). A critical step
in metastasis: in vivo analysis of intravasation at the primary tumor. Cancer Res. 60,
Wyckoff, J. B., Wang, Y., Lin, E. Y., Li, J. F., Goswami, S., Stanley, E. R., Segall, J.
E., Pollard, J. W. and Condeelis, J. (2007). Direct visualization of macrophage-
assisted tumor cell intravasation in mammary tumors. Cancer Res. 67, 2649-2656.
Xue, C., Wyckoff, J., Liang, F., Sidani, M., Violini, S., Tsai, K. L., Zhang, Z. Y., Sahai,
E., Condeelis, J. and Segall, J. E. (2006). Epidermal growth factor receptor
overexpression results in increased tumor cell motility in vivo coordinately with
enhanced intravasation and metastasis. Cancer Res. 66, 192-197.
Yamaguchi, H., Pixley, F. and Condeelis, J. (2006). Invadopodia and podosomes in
tumor invasion. Eur. J. Cell Biol. 85, 213-218.
Zerbe, L. K., Dwyer-Nield, L. D., Fritz, J. M., Redente, E. F., Shroyer, R. J., Conklin,
E., Kane, S., Tucker, C., Eckhardt, S. G., Gustafson, D. L. et al. (2008). Inhibition
by erlotinib of primary lung adenocarcinoma at an early stage in male mice. Cancer
Chemother. Pharmacol. 62, 605-620.
Journal of Cell Science