Cell Reports

Report

Wiskott-Aldrich Syndrome Protein RegulatesLeukocyte-Dependent Breast Cancer MetastasisDan Ishihara,1,5 Athanassios Dovas,1,5 Lorena Hernandez,1 Maria Pozzuto,2 Jeffrey Wyckoff,1,3 Jeffrey E. Segall,1,3

John S. Condeelis,1,3 Anne R. Bresnick,2 and Dianne Cox1,3,4,*1Department of Anatomy and Structural Biology2Department of Biochemistry3Gruss Lipper Biophotonics Center4Department of Developmental and Molecular BiologyAlbert Einstein College of Medicine, Bronx, NY 10461, USA5These authors contributed equally to this work

*Correspondence: dianne.cox@einstein.yu.eduhttp://dx.doi.org/10.1016/j.celrep.2013.07.007

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,

distribution, and reproduction in any medium, provided the original author and source are credited.

SUMMARY

A paracrine interaction between epidermal growthfactor (EGF)-secreting tumor-associated macro-phages (TAMs) and colony-stimulating factor 1(CSF-1)-secreting breast carcinoma cells promotesinvasion and metastasis. Here, we show that micedeficient in the hematopoietic-cell-specific Wiskott-Aldrich syndrome protein (WASp) are unable tosupport TAM-dependent carcinoma cell invasionand metastasis in both orthotopic and transgenicmodels of mammary tumorigenesis. Motility andinvasion defects of tumor cells were recapitulatedex vivo upon coculture with WASp�/� macrophages.Mechanistically, WASp is required for macrophagesto migrate toward CSF-1-producing carcinomacells, as well as for the release of EGF throughmetalloprotease-dependent shedding of EGF fromthe cell surface of macrophages. Our findings sug-gest that WASp acts to support both the migrationof TAMs and the production of EGF, which in concertpromote breast tumor metastasis.

INTRODUCTION

Cancer progression is a complex, multistep process consisting

of neoplastic cell transformation, tumor growth, invasion, and

metastasis. Although metastasis is the major cause of cancer

deaths, it remains poorly characterized (Valastyan and Wein-

berg, 2011). Malignant dissemination is not an exclusively

cancer-cell-autonomous process; other constituents of the

tumor microenvironment, such as inflammatory cells, heavily

influence cancer progression (Hanahan and Coussens, 2012).

Additionally, clinical studies on invasive breast cancer have

demonstrated that tumor-associatedmacrophage (TAM) density

correlates with poor prognosis (Qian and Pollard, 2010).

Indeed, TAMs promote angiogenesis and extracellular matrix

proteolysis, and enhance tumor cell invasion and dissemination

(Joyce and Pollard, 2009).

Circulating levels of colony-stimulating factor 1 (CSF-1), a

pleiotropic macrophage growth factor and chemoattractant,

correlate with poor prognosis in breast cancer patients, and

high levels of CSF-1 are found in invasive, but not in situ, breast

cancer (Qian and Pollard, 2010). In agreement with these

observations, CSF-1 deficiency in the polyoma middle-T antigen

mouse model of mammary carcinogenesis (MMTV-PyMT) led to

a reduction in TAM density, delayed the development of tumors

to invasive metastatic carcinoma, and reduced lung metastasis

with no change in primary tumor growth (Lin et al., 2001), demon-

strating that CSF-1 played a critical role in breast tumor progres-

sion and metastasis.

Macrophages and breast carcinoma cells depend on an

epidermal growth factor (EGF)/CSF-1 paracrine loop for invasion

both in vitro (Goswami et al., 2005) and in vivo (Wyckoff et al.,

2004). In vivo tumor cell invasion was blocked by inhibiting either

the EGF receptor (EGFR) or the CSF-1 receptor (CSF-1R),

expressed by breast carcinoma cells and macrophages, res-

pectively (Wyckoff et al., 2004). Given that recruitment of higher

numbers of TAMs has been reported in EGFR-positive breast

tumors (Leek et al., 2000), blockade of the paracrine interaction

may have therapeutic benefit in the treatment of breast cancer.

Additionally, targeting TAMs is attractive because, unlike cancer

cells, they are genetically stable and less likely to develop drug

resistance.

We demonstrate here that Wiskott-Aldrich syndrome

protein (WASp) could be a potential therapeutic target.

Wiskott-Aldrich syndrome (WAS) is an X-linked genetic

disorder resulting from mutations in the WAS gene (Thrasher

and Burns, 2010). WASp is expressed exclusively in none-

rythroid hematopoietic cells, unlike its ubiquitously expressed

homolog, neural (N)-WASP. WASp is an actin nucleation-

promoting factor that regulates chemotaxis (Takenawa and

Suetsugu, 2007). Since macrophages of WAS patients are

defective in chemotaxis toward CSF-1 (Zicha et al., 1998),

we hypothesized that WASp deficiency may block the para-

crine interaction between TAMs and breast carcinoma cells,

Cell Reports 4, 429–436, August 15, 2013 ª2013 The Authors 429

Figure 1. WASp Is Required for Cell Elongation and Invasion of Both Breast Carcinoma Cells and Macrophages

MTLn3GFP cells were cultured alone or with WASp+/+, WASp�/�, or WASp�/� rescued (WASp-Re) BMMs for 16 hr.

(A) Representative phase-contrast images of the indicated conditions. MTLn3 cells are denoted by a C in coculture images. Scale bar: 10 mm.

(B and C) Quantification of the average elongation of (B) MTLn3GFP and (C) the indicated macrophage types. n R 4 independent experiments. *p < 0.05,

**p < 0.01 compared with control cells.

(D and E) Quantification of the fraction of MTLn3 (D) or BMMs (E) invading above 20 mm in the absence (�) or presence of WASp+/+ or WASp�/� BMMs from three

independent mice.

Error bars signify SEM from five independent fields. ****p < 0.0001 compared with MTLn3 cultured conditions with WASp+/+ BMMs. See also Figure S1 and

Movies S1 and S2.

disabling their comigration and attenuating metastatic

dissemination.

RESULTS

Macrophage WASp Is Required for Breast CarcinomaCell Interaction In VitroTo test whether WASp plays a role in the EGF/CSF-1 paracrine

interaction, we cocultured bone-marrow-derived macrophages

(BMMs) from WASp+/+ or WASp�/� mice with GFP-expressing

MTLn3 cells (MTLn3GFP). Contrary to the WASp�/� BMMs,

the WASp+/+ BMMs induced an increase in MTLn3GFP elonga-

tion (Figures 1A and 1B), indicative of the acquisition of a motile

phenotype (Goswami et al., 2005). Concurrently, the WASp+/+

BMMs elongated in the presence of MTLn3GFP cells, whereas

the WASp�/� BMMs did not (Figures 1A and 1C). Expression

of human WASp in WASp�/� BMMs restored both MTLn3 and

BMM elongation in cocultures (Figures 1B and 1C). Live-cell

imaging of BMMs added to MTLn3GFP cultures showed that

within the first few hours of coculture, the WASp+/+ BMMs elon-

gated robustly toward MTLn3GFP cells, whereas the WASp�/�

BMMs did not (Figures S1A–S1C; Movies S1 and S2).

To test whether WASp was required for macrophage-depen-

dent MTLn3 cell invasion into 3D matrices, we overlaid BMM

and MTLn3GFP cocultures with a collagen I gel and determined

the fraction of invading cells (Goswami et al., 2005). WASp+/+

430 Cell Reports 4, 429–436, August 15, 2013 ª2013 The Authors

BMMs enhanced MTLn3GFP invasion into the matrix 7-fold,

whereas MTLn3GFP invasion was not enhanced by WASp�/�

BMMs (Figures 2A, 2B, and S1D). In the presence of MTLn3GFP

cells, the fraction of coinvading WASp+/+ BMMs was increased

significantly compared with WASp�/� BMMs (Figures 2A, 2C,

and S1D). WASp expression was therefore necessary for

BMMs to adopt a migratory phenotype and to engage in and

maintain the paracrine interaction with breast carcinoma cells,

and for BMMs and breast carcinoma cells to coinvade a three-

dimensional (3D) collagen matrix. Importantly, WASp was not

required for BMM invasion into a fibrous collagen matrix in the

absence of carcinoma cells (A.D. Haein Park, I. Maridonneau-

Parini, and D.C., unpublished data), which suggests that WASp

is required for movement in response to a soluble guidance

cue (in this case, CSF-1 provided by carcinoma cells). This is

consistent with the well-established role of WASp in the chemo-

taxis of macrophages. These results prompted us to investigate

whether WASp is required for macrophage-dependent breast

tumor invasiveness and metastasis in mouse models of breast

cancer.

WASp Is Required for Efficient Breast CancerMetastasisTo specifically test the effect of myeloid WASp on tumor inva-

siveness and metastasis, we crossed WASp+/� or WASp�/�

mice with Rag2�/� mice to generate xenograft-competent

Figure 2. WASp Is Required for TAM-Dependent Breast Carcinoma

Cell Metastasis, but Not Primary Tumor Growth, in a Rat Orthotopic

Breast Tumor Model

(A) PrimaryMTLn3-Cerulean tumor growth was determined by the average day

of tumor detection after injection, age of primary tumors, and volume of

primary tumors upon harvest.

(B) Representative fluorescent images of spontaneous lung metastases. Scale

bar: 10 mm.

(C) Quantification of spontaneous lung metastases: number of metastatic

nodules (<5 cells in cluster; left graph) or total carcinoma cell number from the

whole lung (right graph). n = 9 WASp+/� and 11 WASp�/� tumor-bearing mice.

*p < 0.05, **p < 0.01 by two-tailed Student’s t test and Mann-Whitney

significance test.

Error bars signify SEM. See also Figure S2.

mice that lack mature and functional B and T lymphocytes, and

injected cyan-fluorescent MTLn3 cells (MTLn3-Cerulean) into

the mammary glands. The time of initial tumor detection after

injection, tumor age, and volume upon harvest did not differ

between WASp+/� and WASp�/� tumor-bearing mice (Fig-

ure 2A). However, lung metastasis, measured as metastatic

nodules or total number of carcinoma cells per whole lung,

was significantly reduced in WASp�/� mice compared with

WASp+/� mice (Figures 2B and 2C). These data suggested that

host WASp expression was required for efficient lung metastasis

of breast tumor cells, but not for primary tumor growth.

We confirmed the above data using the triple-negative human

breast carcinoma cell line MDA-MB-231. In vitro MDA-MB-231

cell invasion was significantly reduced in the presence of

WASp�/� BMMs (Figure S2A), whereas WASp+/� and WASp+/+

BMMs promoted MDA-MB-231 cell invasion equally. Similar

results were obtained using the highly invasive subline MDA-

MB-231-4173, characterized for its lung tropism in metastasis

(Figure S2B). Likewise, orthotopic tumors of MDA-MB-231-

4173 Cerulean cells in Rag2�/�WASp+/� or Rag2�/�WASp�/�

mice resulted in fewer lung metastases in WASp�/� mice

compared with WASp+/� mice despite equal growth parameters

(Figures S2C and S2D).

We also used the immune-competent MMTV-PyMT trans-

genic mouse breast cancer model, which resembles human

breast cancer progression (Lin et al., 2003). No significant differ-

ences in primary tumor growth were detected between

PyMT+WASp+/� and PyMT+WASp�/� mice (Figure S2E). Semi-

quantitative RT-PCR of PyMT messenger RNA (mRNA) was

performed to determine the extent of metastasis in the lung

(Lin et al., 2001), and the results revealed significantly more

PyMT mRNA levels in the lungs of WASp+/� mice compared

with WASp�/� mice (Figure S2F). Histologic evaluations of

hematoxylin and eosin (H&E)-stained lungs of PyMT tumors

showed more metastatic nodules in PyMT+WASp+/� mice

compared with PyMT+WASp�/� mice (Figure S2G).

Crucially, none of the tumor cells examined expressed WASp

(Figure S2H). We have thus shown in three different breast tumor

models (a rat, a human xenograft, and a transgenic model of

mammary carcinogenesis) that leukocyte-restricted WASp was

required for efficient metastasis to the lung.

WASp Is Required for TAM-Mediated Breast CarcinomaCell Motility, Invasion, and Intravasation In VivoBecause WASp is required for macrophage chemotaxis, it was

possible that WASp deficiency affected TAM recruitment. To

address this concern, we examined the TAM content of primary

tumors by intravital imaging of MTLn3-Cerulean tumors from

Rag2�/�WASp+/� or WASp�/� mice, with TAMs identified by

the uptake of Texas red-dextran (Wyckoff et al., 2007). MTLn3-

Cerulean tumors from WASp�/� mice contained slightly lower

numbers of TAMs (Figure 3A), especially at the tumor cortex;

however, these differences were not statistically significant.

These results suggested that WASp did not play a major role in

the recruitment of macrophages to late-stage carcinomas and

thus could not account for the dramatic reduction in tumor

metastasis.

It has been shown that TAMs promote the motility of tumor

cells that are in close proximity (Roussos et al., 2011; Wyckoff

et al., 2007). Multiphoton intravital imaging showed decreased

TAM-proximal tumor cell motility throughout the tumors of

WASp�/� mice compared with those of WASp+/� mice (Fig-

ure 3B; Movies S3 and S4). Therefore, despite the slightly lower

numbers of TAMs in WASp�/� mice at the tumor cortex, carci-

noma cell motility throughout the tumor, even in areas of similar

TAM density, was significantly reduced. To directly test whether

WASp in TAMs was required for the paracrine interaction with

breast tumor cells in vivo, we employed an in vivo invasion assay

(Ojalvo et al., 2010; Patsialou et al., 2012; Wyckoff et al., 2000)

in which collection needles containing Matrigel and either

EGF or buffer were inserted into MTLn3-Cerulean tumors of

Cell Reports 4, 429–436, August 15, 2013 ª2013 The Authors 431

Figure 3. WASp Is Required for TAM-Dependent Carcinoma Cell Motility, Invasion, and Intravasation In Vivo

(A) The number of Texas-red dextran-positive TAMs from WASp+/� and WASp�/� mice was quantified 24 hr after Texas-red dextran injection. Images were

acquired at 5 mm steps starting from the tumor cortex to a final depth of 100 mm using intravital two-photon microscopy.

(B) Quantification of the average number of motile MTLn3 cells per field determined by intravital imaging. n > 3 mice per genotype. *p < 0.05 using the Mann-

Whitney significance test. See also Movies S3 and S4.

(C) Total number of invasive cells collected intomicroneedles containing either 25 nMEGF or buffer. n = 4mice per genotype. **p < 0.01 Student’s t test compared

with buffer-containing needles in WASp+/� mice.

(D) Tumor cell blood burden in WASp+/� or WASp�/� mice bearing MTLn3-derived breast tumor following 3 weeks of tumor growth. Graphs show the average

number of cells (left) and colonies (right) per milliliter of blood. n > 4 mice per genotype. Error bars signify SEM. **p < 0.05.

See also Figure S2.

Rag2�/�WASp+/� and WASp�/� mice. The EGF-filled needles

collected significantly more MTLn3-Cerulean cells than the

buffer-filled needles from tumors of WASp+/� mice (Figure 3C).

In contrast, there was no EGF-induced in vivo invasion from

tumors of WASp�/� mice even though TAM levels were only

reduced by a maximum of 30%.

TAMs also assist tumor cell intravasation (Wyckoff et al.,

2007). Consistently, blood from WASp�/� mice contained fewer

viable circulating tumor cells compared with blood from

WASp+/� mice (Figure 3D), suggesting that breast tumor cell

intravasation was reduced in WASp�/� mice. Overall, these

data support a role of host WASp in tumor cell motility, invasion,

and intravasation at the primary tumor site.

WASp Is Required for EGF SheddingThe inability ofWASp�/�mice to support carcinoma cell invasion

prompted us to further examine the mechanisms by which

WASp contributed to the paracrine interaction with carcinoma

cells. Apart from motility, WASp regulates secretory processes

(Thrasher and Burns, 2010). Given the central role of TAM-

secreted EGF in the paracrine loop, we explored EGF secretion

432 Cell Reports 4, 429–436, August 15, 2013 ª2013 The Authors

in BMMs. Conditioned media from WASp+/+ BMMs promoted

MTLn3GFP 3D invasion, whereas conditioned media from

WASp�/� BMM did not (Figure 4A). Neutralizing antibodies

against EGF attenuated MTLn3GFP cell invasion by the condi-

tioned media (Figure 4B). Consistently, addition of purified EGF

to conditioned media from WASp�/� BMMs rescued MTLn3

cell invasion (Figure 4C). Collectively, these results demon-

strated that EGF was necessary and sufficient to promote

MTLn3 invasion in vitro, and suggested that WASp was required

for EGF secretion.

However, EGF mRNA and proEGF protein levels were equal

between WASp+/+ and WASp�/� BMMs under basal conditions

and were upregulated to the same extent following CSF-1 treat-

ment (Figure S3). This prompted us to investigate macrophage

EGF trafficking and shedding. We used a well-established

reporter of EGF shedding in which alkaline phosphatase (AP)

replaces part of the extracellular portion of proEGF (APEGF)

(Sahin et al., 2006). Infection of primary BMMs with this reporter

proved inefficient for a consistent quantitative assay (data not

shown), so we assayed previously characterized macrophage

cell lines (RAW/LR5) infected with a nontargeting, control small

Figure 4. WASp Is Required for EGF Release

(A) Quantification of in vitro 3D invasion using MTLn3GFP cells cultured alone (�) or with conditioned media (CM) from WASp+/+ or WASp�/� BMMs.

(B) Quantification of 3D invasion of MTLn3GFP cells in the absence (�) or presence of WASp+/+ CM with either a nonspecific immunoglobulin G (IgG) or two

different EGF-neutralizing antibodies (aEGF Ab1 or Ab2).

(C) Addition of exogenous EGF (5 nM) to WASp�/� BMM CM restores invasion of MTLn3GFP cells. n R 3 experiments per condition.

(D) Confocal sections of control sh and shWASp RAW/LR5 cells expressing APEGF-sfGFP. Unpermeabilized fixed cells were stained with an antibody against

extracellular AP. Scale bar: 10 mm.

(E) Equal expression levels of APEGF-sfGFP (arrow) expressed by the control sh and shWASp cells, detected with an antibody against GFP.

(F) CM from the indicated APEGF-sfGFP-expressing cells under the conditions shown were collected and assayed for shed AP activity.

(G) Quantification of MTLn3GFP invasion with CM collected from WASp�/� BMM infected with either a control vector or a vector expressing recombinant

soluble EGF.

Error bars signify SEM. *p < 0.05, **p < 0.01 and ***p < 0.001. See also Figures S3 and S4.

Cell Reports 4, 429–436, August 15, 2013 ª2013 The Authors 433

hairpin RNA (shRNA, hereafter termed control sh) or one that

targets WASp (shWASp) (Dovas et al., 2009; Figure S4A). Similar

to BMMs, these cells engaged in WASp-dependent paracrine

interactions with carcinoma cells (Figures S4B–S4D). Control

sh or shWASp cells were infected with a retrovirus expressing

APEGF fused to superfolder GFP in its cytoplasmic tail

(APEGF-sfGFP). Both control sh and shWASp cells were able

to traffic APEGF-sfGFP to the cell surface, with no observable

differences in subcellular distribution (Figure 4D). Despite equal

expression levels of APEGF-sfGFP (Figure 4E) and basal

shedding of AP activity, the shWASp cells shed significantly

lower amounts of APEGF-sfGFP than the control sh cells

following CSF-1 stimulation (Figure 4F). Treatment of cells with

the broad-spectrum inhibitor GM6001 reduced shedding to its

basal levels (Figure 4F), indicating that WASp regulates CSF-1-

mediated EGF shedding in a metalloprotease-dependent

manner. Consistently, media from WASp�/� BMMs infected

with a construct expressing a secretable form of the mature,

soluble human EGF (Yao et al., 1998) bypassed the require-

ment for EGF shedding and promoted invasion of MTLn3 cells

(Figure 4G).

DISCUSSION

We have demonstrated that WASp regulated macrophage-

mediated tumor cell invasion in vitro and tumor cell motility,

invasion, intravasation, and lung metastasis, but not tumor

growth, in vivo. These results corroborate previous studies

implicating CSF-1 and TAMs in metastasis and suggest that

inhibition of effectors downstream of CSF-1R signaling may be

an effective strategy for antimetastatic therapy.

Additionally, we uncovered a role for WASp in the regulation of

CSF-1R-mediated EGF shedding, but found that EGF mRNA,

protein levels, and cell-surface expression were WASp indepen-

dent. Our data suggest that WASp may bridge CSF-1R signaling

to a cell-surface metalloprotease activity. Importantly, dendritic

cells deficient in WASp interacting protein (WIP), which binds

and regulates WASp levels in vivo, showed reduced secretion

of proMMP2 and proMMP9 (Banon-Rodrıguez et al., 2011).

Interference with the WASp pathway in myeloid cells may thus

lead to systemic defects in pericellular proteolysis.

WASp may regulate EGF shedding from the cell surface via

several, perhaps interdependent, mechanisms: (1) directly via

delivery of vesicles containing the sheddase(s) by F-actin-

mediated propulsion, (2) indirectly via the establishment of cell

polarity and the formation of F-actin conduits for sheddase

delivery, and (3) indirectly via the formation of podosomes,

F-actin-rich structures that concentrate proteolytic activities.

Such mechanisms have been proposed to act in the delivery of

proteases to the plasma membrane and to invadopodia of inva-

sive tumor cells (Poincloux et al., 2009). Further experiments will

be needed to determine the exact mechanism by which WASp

regulates EGF shedding.

The decreased in vivo cell invasion observed inWASp�/�mice

despite the presence of exogenous EGF supplied through the

microneedle (Figure 3C) may appear to contradict the in vitro

data indicating that EGF alone restored breast carcinoma cell in-

vasion (Figure 4C). However, efficient chemotaxis in the tumor

434 Cell Reports 4, 429–436, August 15, 2013 ª2013 The Authors

requires the propagation of chemotactic signals from cell to

cell in a streaming fashion (Roussos et al., 2011; Sharma et al.,

2012) so as to maintain the migration of cells. Consistent with

this, blockade of CSF-1R in the presence of EGF was equally

effective as EGFR blockade in reducing the number of invasive

cells collected (Wyckoff et al., 2004). Given that WASp was

required for the elongation of BMMs toward tumor cells in vitro

(Figure 1), our data suggest that WASp in macrophages contrib-

utes to carcinoma cell invasion by supporting both macrophage

chemotaxis and EGF release.

Our findings suggest thatWASp inhibition may have therapeu-

tic benefit in breast tumors. We argue that inhibition of a protein

downstream of CSF-1R could bypass several issues associated

with CSF-1R blockade, such as the nonspecificity of kinase

inhibitors or elevated levels of circulating CSF-1 in the case of

anti-CSF-1R antibodies (Hume andMacDonald, 2012). Addition-

ally, WASp inhibition could affect the functions of other hemato-

poietic cells that play a role in metastasis, such as IL-4-secreting

CD4+ T cells that stimulate EGF production by TAMs (DeNardo

et al., 2009) and platelets, which are important for tumor cell

seeding at the metastatic site (Gay and Felding-Habermann,

2011). While we cannot completely rule out the contributions of

other hematopoietic cells in our system, we argue that TAMs

play the most critical roles in the functions tested. Indeed,

specific ablation of TAMs by genetic deletion of CSF-1 (Lin

et al., 2001), administration of liposome-encapsulated clodro-

nate, or inhibition of CSF-1R (Hernandez et al., 2009a; Patsialou

et al., 2009;Wyckoff et al., 2004) recapitulates all of our observed

long- and short-term phenotypes. In addition, WASp deficiency

caused defects in tumor cell invasion in the primary tumor prior

to entry of tumor cells into the circulation and seeding at

secondary sites, steps where other leukocytes may contribute.

Therefore, the defects observed in the absence of WASp can

be largely attributed to defects in TAMs. Importantly, TAM-

specific delivery of pharmacological agents is feasible (Leusch-

ner et al., 2011). In this respect, WASp could be selectively

targeted in TAMs and not other leukocytes.

WASpmay be a viable candidate because we have shown that

although WASp is required for TAMs to promote breast carci-

noma cell invasion and metastasis, it is expressed exclusively

in genetically stable hematopoietic cells. In support of our re-

sults, an Oncomine analysis demonstrated that WASp was

significantly overexpressed in high-grade (p < 1.1 3 10�6) and

triple-negative (p < 1.6 3 10�8) tumors but was significantly

underexpressed in estrogen receptor (ER)-positive tumors (p <

5.3 3 10�14). This finding agrees with a study showing that

inflammatory leukocyte infiltration correlates with the aggres-

siveness of ER-negative breast carcinomas (Chavey et al.,

2007). The identification and characterization of host factors

that play an essential role in tumor progression may ultimately

lead to new therapeutic targets and expand the available options

for cancer therapy.

EXPERIMENTAL PROCEDURES

Mice

All procedures involving mice were conducted in accordance with National

Institutes of Health regulations concerning the use and care of experimental

animals. The Institutional Animal Use Committee of Albert Einstein College of

Medicine approved the use of mice in this study.

In Vivo Experiments

In vivo experiments, including intravital microscopy, in vivo invasion, intravasa-

tion, and lung metastasis, are described in the Extended Experimental

Procedures.

In Vitro Experiments

In vitro experiments, including 2D and 3D coculture assays, AP assay, and

confocal microscopy, are described in the Extended Experimental

Procedures.

Data Analysis

Results were analyzed by two-tailed Student’s t test. Nonparametric statistical

analysis by two-tailedMann-Whitney significance test was performed for anal-

ysis of lungmetastasis inMTLn3-Cerulean-injectedmice and intravital imaging

analysis; a p value of < 0.05 was considered significant.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Extended Experimental Procedures, four

figures, and four movies and can be found with this article online at http://

dx.doi.org/10.1016/j.celrep.2013.07.007.

ACKNOWLEDGMENTS

We thank the researchers for providing reagents as indicated. We thank

members of the Condeelis, Segall, Hogdson, and Stanley laboratories for

helpful comments and discussions, and the staffs of the Analytical Imaging

Facility and Animal Housing Facility of Einstein College of Medicine for tech-

nical assistance. This work was supported by National Institutes of Health

grants GM071828 (D.C., A.D., and D.I.), CA100324 (A.D., A.R.B., J.S., J.C.,

and D.C.), and CA110269 (L.H.).

Received: March 11, 2013

Revised: May 24, 2013

Accepted: July 8, 2013

Published: August 1, 2013

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