Epidermal Growth Factor Receptor Overexpression Results in Increased Tumor Cell Motility In vivo Coordinately with Enhanced Intravasation and Metastasis

2006;66:192-197. Cancer Res   Chengsen Xue, Jeffrey Wyckoff, Fubo Liang, et al.   Enhanced Intravasation and Metastasis

Coordinately withIn vivoin Increased Tumor Cell Motility Epidermal Growth Factor Receptor Overexpression Results

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Epidermal Growth Factor Receptor Overexpression Results in

Increased Tumor Cell Motility In vivo Coordinately with

Enhanced Intravasation and Metastasis

Chengsen Xue,1Jeffrey Wyckoff,

1,3Fubo Liang,

2Mazen Sidani,

1Stefania Violini,

1Kun-Lin Tsai,

1

Zhong-Yin Zhang,2Erik Sahai,

1John Condeelis,

1,3and Jeffrey E. Segall

1

Departments of 1Anatomy and Structural Biology and 2Molecular Pharmacology and 3Analytical Imaging Facility,Albert Einstein College of Medicine, Bronx, New York

Abstract

Although overexpression of the epidermal growth factorreceptor (EGFR; ErbB1) has been correlated with poorprognosis in breast and other cancers, clinical trials of ErbB1inhibitors have shown limited efficacy in inhibiting tumorproliferation. To evaluate other possible roles of ErbB1 intumor malignancy besides proliferation, we have developed aseries of tools for analysis of intravasation. Overexpression ofErbB1 in MTLn3 mammary adenocarcinoma cells results inincreased intravasation and lung metastasis from tumorsformed by injection of cells in the mammary fat pad. However,increased ErbB1 expression has no effect on primary tumorgrowth and lung seeding efficiency of cells injected i.v.Chemotactic responses to low concentrations of EGF in vitroand cell motility in vivo in the primary tumor measured usingintravital imaging are significantly increased by ErbB1 over-expression. The increased cell motility is restricted to ErbB1-overexpressing cells in tumors containing mixtures of cellsexpressing different ErbB1 levels, arguing for a cell-autonomouseffect of increased ErbB1 expression rather than alterationof the tumor microenvironment. In summary, we proposethat ErbB1 overexpression makes more significant contribu-tions to intravasation than growth in some tumors andpresent a novel model for studying ErbB1 contributions totumor metastasis via chemotaxis and intravasation. (Cancer Res2006; 66(1): 192-7)

Introduction

Metastasis involves multiple steps, including growth of aprimary tumor, intravasation, and arrest and growth in secondarysites. The steps of growth of the primary tumor, as well as arrestand growth in secondary sites have been extensively studied, withgenes affecting these steps being identified and studied (1–5).Studies of intravasation have lagged due to limitations intechniques for analyzing this step. The requirements for studyof intravasation include quantitation of the cell number in thecirculation, the ability to observe cells in the primary tumor, andidentification of cell lines in which intravasation is selectivelyaltered (6).

The epidermal growth factor receptor (EGFR; also referred to asErbB1) is often overexpressed in breast and other cancers (7–9).It has been correlated with poor prognosis (10, 11), and theseobservations have stimulated the development of ErbB1 inhibitors(12). Both antibody inhibitors, such as cetuximab, and smallmolecule inhibitors, such as gefitinib and Tarceva, have beendeveloped to inhibit ErbB1 (13–15). Clinical trials of these ErbB1inhibitors have shown some but much more limited efficacy inblocking tumor growth than would be expected based on theproportions of tumors with overexpressed ErbB1 (16, 17). Theseresults raise the possibility that ErbB1 might contribute to themalignant potential of tumors through affecting other character-istics besides just proliferation. ErbB1 can increase in vitro tumorcell motility and invasion (18–23). However, the in vivo contribu-tion of the increased motility and invasion, which is critical tounderstanding metastasis of tumors, is still not clear.We have developed methods for evaluating cell properties at the

primary tumor, including multiphoton imaging and quantitation oftumor cell number in the blood (6, 24–28). Multiphoton imagingof green fluorescent protein (GFP)–labeled primary tumors enablesdirect visualization of tumor cell behavior in vivo . Quantitation oftumor cell number in the blood is a direct evaluation of theefficiency of intravasation. The combination of these two measure-ments allows identification of changes in cell movement that cancontribute to intravasation efficiency. Using MTLn3 and MTC ratmammary adenocarcinoma cell lines, we showed that there can besignificant differences in intravasation between metastatic andnonmetastatic cells. More specifically, the metastatic MTLn3cells showed greater orientation toward blood vessels within theprimary tumor, whereas nonmetastatic MTC cells fragmentedwhen interacting with vessels (6, 24). The findings illustrate thevalue of a direct visualization of cell properties in vivo fordissection of the metastatic process.In this article, we provide direct in vivo evidence for contributions

of ErbB1 to metastasis independent of effects on proliferation.Overexpression of ErbB1 in MTLn3 rat mammary tumor cellsresults in significantly increased intravasation and metastasis fromthe primary tumor, without any changes in primary tumor growth.Chemotactic efficiency in vitro and tumor cell motility in vivo werealso significantly increased. Increased ErbB1 expression did notaffect lung seeding efficiency of cells injected i.v. Our studies areconsistent with in vitro and in vivo studies showing that ErbB1expression can enhance invasiveness (18–23).

Materials and Methods

Cell culture and establishing EGFR transfectants in MTLn3 ratmammary adenocarcinoma cells. Rat mammary adenocarcinoma cell

line MTLn3 was obtained from Dr. Garth Nicolson (Institute for Molecular

Note: E. Sahai is currently at the Tumour Cell Biology Laboratory, Cancer ResearchUnited Kingdom, London Research Institute, 44 Lincoln’s Inn Fields, London WC2A3PX, United Kingdom.

Requests for reprints: Jeffrey E. Segall, Department of Anatomy and StructuralBiology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY10461. Phone: 718-430-4237; Fax: 718-430-8996; E-mail: [email protected].

I2006 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-05-1242

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Research Article

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Medicine, Huntington Beach, CA; ref. 29) and was maintained in aMEM(Life Technologies, Inc., Gaithersburg, MD) supplemented with 5% fetal

bovine serum and penicillin-streptomycin (Life Technologies). For trans-

fection assays, MTLn3 cells were seeded in six-well plates and grown until

70% confluent. These cells were subjected to retroviral transduction ofconstructs containing the retroviral expression vector pLXSN alone or

pLXSN driving expression of ErbB1 (ref. 30; provided by Dr. David Stern,

Yale University). The vectors were packaged in the Phoenix cell line

following standard protocols (31), and geneticin-resistant clones wereisolated. Cell growth rate and morphology of transductants was determined

to be the same as parental cells, and the expression of ErbB1 was confirmed

by flow cytometry and Western blot.

Microchemotaxis chamber assay. A 48-well microchemotaxis chamber(Neuroprobe, Cabin John, MD) was used as previously described (32), except

that L15 containing 0.35% bovine serum albumin was used instead of

aMEMH. For measurement of migration in response to EGF (LifeTechnologies Bethesda Research Laboratories, Gaithersburg, MD), filters

were coated with 27 Ag/mL rat tail collagen (BD Biosciences, Palo Alto, CA).

After inserting the filters in the chamber, 20,000 cells detached by trypsin/

EDTA were plated into the wells of the upper chamber. The chambers wereincubated for 3 hours at 37jC before counting the number of cells crossing

the filter.

To evaluate the in vitro cell-autonomous chemotactic efficiency ofMTLn3-

B1 cells compared with MTLn3-PL cells, mixtures of equal numbers offluorescent MTLn3-B1-GFP and nonfluorescent MTLn3-PL cells were used in

the 48-well chemotaxis assay as described above. After fixation, the filters

were stained with.5 mg/mL 4V,6-diamidino-2-phenylindole (DAPI) for 2minutes, and then images of DAPI and GFP fluorescence were taken using a�10 objective. In each experiment, images of unscrapedwells were also taken

to determine the actual relative percentage of cells plated per experiment,

and this information was used in calculating the relative chemotacticefficiency. ImageJ (developed by Wayne Rasband at the NIH, Bethesda, MD;

http://rsb.info.nih.gov/ij/, 1997-2005) macros were written to identify all cells

using the DAPI image and MTLn3-B1 cells by GFP fluorescence. For each

condition, the relative proportion of MTLn3-B1 cells was determined andused to calculate the relative chemotactic efficiency of MTLn3-B1 cells

compared with MTLn3-PL cells. Iressa (AstraZeneca, Manchester, England)

and AG879 (SigmaT2067) were used to inhibit ErbB1 and ErbB2, respectively.Immunoblotting. Cells were grown to 70% confluency in a 10-cm cell-

culturing dish and then incubated with serum-free medium for 4 hours. The

medium was changed to fresh serum-free medium with or without 5 nmol/L

EGF, and cells were incubated for 30 seconds. Cells were then washed with

cold PBS and lysed in 0.5 mL lysis buffer [50 mmol/L Tris-HCl (pH 7.5),

1% Triton X-100, 5 mmol/L EGTA, 1 mmol/L EDTA, 150 mmol/L NaCl,

10% glycerol, 1 mmol/L phenylmethylsulfonyl fluoride, 50 mmol/L sodium

fluoride, Complete Protease Inhibitor Cocktail (Roche Diagnostics Corp.,

Indianapolis, IN)]. The plates were scraped with a rubber policeman and

incubated on ice for 20 minutes. The lysate was precleared by centrifugation

at 15,000 rpm for 15 minutes. Lysate protein concentration was measured

using bicinchoninic acid protein assay reagent (Pierce, Rockford, IL).

Protein (20 Ag) of each sample was loaded and separated by SDS-PAGE and

transferred electrophoretically to nitrocellulose membranes, which were

immunoblotted by appropriate antibodies followed by incubation with

horseradish peroxidase–conjugated secondary antibodies. The following

antibodies were used: anti-h-actin (Sigma, St. Louis, MO), anti-phosphotyr-

osine (PY20, BD Biosciences, San Diego, CA), anti-phospho-ErbB1 (Tyr1173,

Cell Signaling Technology, Beverly, MA), anti-phospho-ErbB2 (Tyr877, Cell

Signaling Technology), and anti-ErbB2 monoclonal (NeoMarkers, Fremont,

CA). The blots were developed by using SuperSignal West Pico

Chemiluminescent Substrate (Pierce).

Spontaneous and experimental metastasis assays. All animal studies

described here were done according to protocols approved by theInstitutional Animal Care and Use Committee of Albert Einstein College

of Medicine. To measure spontaneous metastasis, tumor cells were grown

to 70% to 85% confluence before being harvested for cell counting. Cells(5 � 105) were injected into the right thoracic mammary fat pads of 5- to 7-

week-old female BALB/c severe combined immunodeficient (SCID) mice

(National Cancer Institute, Bethesda, MD). The cells were injected in avolume of 100 AL of PBS with calcium and magnesium through a 25-gauge

needle. Tumor growth rate was monitored at weekly intervals after inoc-

ulation of tumor cells. The tumors were measured in two dimensions, and

tumor volumes were calculated using the formula: (length � width2) / 2.Five weeks after inoculation, the animals were anesthetized, blood burden

was determined as described below followed by sacrifice of the animals and

removal of primary tumors and lungs for histologic analysis as described

below.For the experimental lung metastasis assay, 2.5 � 105 cells were injected

into the lateral tail veins of 5- to 7-week-old female BALB/c SCID mice

(National Cancer Institute). Two weeks after injection, the mice were

euthanized, and the lungs were removed and subjected to histologicexamination as described below.

Measurement of tumor cell blood burden. At the end point of the

spontaneous metastasis assay, mice were anesthetized with Aerrane(Isoflurane, Baxter Pharmaceutical Products, Inc., Deerfield, IL). The right

chest was exposed by a simple skin flap surgery. Blood was taken from the

right atrium via heart puncture with a 25-gauge needle and 1-mL syringe

coated with heparin. Blood (0.2-1.05 mL) was harvested from each animal.The blood was immediately plated into 150-mm-diameter dishes filled with

5% fetal bovine serum/aMEM growth medium. The next day, the plates

were rinsed with fresh medium containing 0.8 mg/mL geneticin to

selectively grow the tumor cells. After 3 to 7 days, all tumor cell clonesin the dish were counted. Tumor blood burden was calculated as total

colonies in the dish divided by the volume of blood taken. To test tumor

cell viability in the blood colony assay process, cultured MTLn3-PL cellswere removed from dishes by 0.25% trypsin/EDTA, and 1,000 tumor cells

were added to fresh mouse blood. The mixtures were then processed as

described above.

Tumor histology and quantitative assessment of the efficiency ofmetastasis. The primary tumors and lungs from each mouse were used for

histologic analysis. Samples were fixed in formalin and embedded in

paraffin, and 5-Am sections were stained with H&E. For each lung sample,

all micrometastases were counted under a light microscope at �10

magnification, and the total lung area was measured using a UMAX

PowerLook III color scanner (UMAX Technologies, Inc., Dallas, TX) and

Adobe Photoshop 5.5 software. Briefly, after scanning lung sections, the

cross-sectional area in pixels was measured using PhotoShop and converted

to mm2 using a calibration factor of 1 pixel = 0.00179 mm2. The efficiency of

lung metastasis then was calculated as metastases per mm2 of lung area.

Intravital imaging by multiphoton microscopy. Tumor imaging was

done as described previously (6, 24, 27, 33). Cells (5 � 105) were injected into

the mammary fat pads of SCID mice as described above and allowed to grow

for 4 weeks. The animal was placed under isoflurane anesthesia, and the

tumor was exposed using a simple skin flap surgery, with as little disruption

of the surrounding vasculature as possible. The animal was then placed in a

30jC temperature chamber on an inverted Olympus IX70 microscope and

imaged using a �20 objective. Briefly, a 10-W Millennium Xs laser (Spectra

Physics, Mountain View, CA) was used to run a Radiance 2000 multiphoton

system (Bio-Rad, Hercules, CA) at 880 nm using a 450/480-nm filter to image

matrix and CFP and a 515/530-nm filter to image CFP and GFP. Time lapse

images of GFP-labeled, MTLn3-PL- and MTLn3-B1-generated tumors were

taken at 60-second intervals for 30 minutes. The images were collected using

Bio-Rad’s Lasersharp 2000 software. During each 1-minute interval, a z series

of 9 to 12 images was taken at a spacing of 10 Am between images, beginning

at the periphery of the tumor and moving into the tumor. For each tumor,

this image acquisition process was repeated for 30 minutes, resulting in a

time lapse three-dimensional z series for analysis of tumor cell motility.

Sites of moving tumor cells in each plane of the z series were identified

by playing the time lapse movie of that plane in ImageJ and marking sites of

movement using a specially written plugin. The total number of movementsites for each plane over the entire 30 minutes was then calculated. The top

four planes (corresponding to the outermost 40 Am of the tumor)

reproducibly provided good enough images for determination of movementsites and were used to calculate the average number of motility sites for

each tumor type.

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Results

Increasing ErbB1 expression in MTLn3 cells significantlyenhances lung metastasis but not tumor size. MTLn3 mam-mary adenocarcinoma cells were transfected with the pLXSNretroviral expression vector containing full-length ErbB1 or withthe empty vector as a control and were designated as MTLn3-B1and MTLn3-PL, respectively. Increased expression of ErbB1 proteinin MTLn3-B1 was confirmed by Western blot (Fig. 1A, left). Asshown in Fig. 1A , expression of other ErbB family members, suchas ErbB2 and ErbB3, was not affected (ErbB4 was not detectable).Fluorescence-activated cell sorting (FACS) analysis confirmedhigher cell surface expression levels of ErbB1 for MTLn3-B1 (datanot shown). No significant changes in morphology or growth ratein vitro were observed for MTLn3-B1 cells compared with MTLn3-PL cells. Stimulation of MTLn3-B1 and MTLn3-PL cells with EGFshowed increased tyrosine phosphorylation of ErbB1 and ErbB2 inMTLn3-B1 cells, indicating that ErbB1/ErbB2 heterodimer signal-ing is increased in these cells (Fig. 1B).To determine the effects of increased ErbB1 expression on tumor

growth and metastasis, the ErbB1 transductants were injected intothe mammary fat pads of SCID mice, and tumor growth wasmonitored. All animals implanted with tumor cells formed tumorsat the site of injection, and the tumors were palpable about 2 weeksafter injection. The dynamics of in vivo tumor growth rates ofMTLn3-B1 and MTLn3-PL were similar. At 5 weeks, the animalswere sacrificed for analysis of spontaneous metastasis efficiency.The mean volumes of MTLn3-pL and MTLn3-B1 tumors were1,825 F 215 and 1,666 F 250 mm3, respectively (Fig. 1C). There areno statistically significant differences in tumor volume (P > 0.05),

indicating that increased ErbB1 expression does not affect tumorgrowth rate of MTLn3 cells.To evaluate the effect of increased ErbB1 on the efficiency of

metastasis, lung samples from the mice were examined physically atautopsy and by light microscopy on H&E-stained paraffin sections.As shown in Fig. 1D , mice bearing MTLn3-B1 tumors hadsignificantly more lung metastases than mice carrying MTLn3-PLtumors. On average, MTLn3-B1 tumors generated 6.00 F 2.59metastases/mm2 compared with 0.49 F 0.26 metastases/mm2

generated by MTLn3-PL tumors. The difference is highly significant(P = 0.0001) and shows that ErbB1 increases spontaneousmetastaticefficiency of MTLn3 cells without affecting primary tumor growth.Increasing tumor cell motility and intravasation by ErbB1

expression. Intravasation is an important metastatic property andcan be evaluated by determining the number of tumor cells presentin blood collected from the right atrium of the heart, beforefiltration by the lungs. The number of viable tumor cells present isthen determined by culturing the blood and counting the numberof tumor cell colonies that form. Using blood added to culturedtumor cells, this assay has anf87% recovery rate. FACS analysis ofblood taken from animals bearing tumors formed by GFP-labeledcells shows a good correlation between the number of coloniespresent on the plate and the number of cells in the blood(correlation coefficient, 0.86). On average, 0.7 mL of blood wastaken from the right atrium via heart puncture from each animalbearing tumors generated by MTLn3-PL and MTLn3-B1. Animalsbearing MTLn3-PL tumors had 37 F 17 tumor cells/mL of blood,whereas animals bearing MTLn3-B1 tumors had 441 F 172 tumorcells/mL blood, a highly significant difference (Fig. 2A ; P = 0.0055).Thus, increased ErbB1 expression can significantly increase intra-vasation rate without affecting primary tumor growth.A mechanism by which increased ErbB1 expression could

enhance intravasation is via motility. Because we have shown thatMTLn3 cells exhibit in vivo invasion in response to gradients of EGF(6), gradients of EGF from blood vessels or stromal cells in tumorscould enhance intravasation. To test whether increasing ErbB1expression enhanced the chemotactic response of MTLn3 cellsto EGF, we used a microchemotaxis chamber migration assay tomeasure chemotactic sensitivity. MTLn3-B1 cells showed strongerresponses at all concentrations of EGF (Fig. 2B), with 5-fold morecells migrating in response to 0.05 and 0.5 nmol/L EGF. Thus,

Figure 1. Increasing ErbB1 expression in MTLn3 cells significantly enhanceslung metastasis but not tumor size. A, Western blot of ErbB1 expression inMTLn3-PL (PL ) and MTLn3-B1 (B1) lines. ErbB2 and ErbB3 expression werealso measured and served as internal controls. B, EGF stimulates both ErbB1and ErbB2 tyrosine phosphorylation in MTLn3-B1 cells. Cells were stimulatedwith 5 nmol/L EGF for 30 seconds, and Western blots were done as described inMaterials and Methods. pErbB1 reflects tyrosine phosphorylation at residue 1173for ErbB1, and pErbB2 represents tyrosine phosphorylation at residue 877 forErbB2. C, primary tumor size at the end point of the spontaneous metastasisassay. MTLn3-PL (16 mice) and MTLn3-B1 (17 mice) cells were injected intothe right mammary fat pads of SCID mice. After 5 weeks, the animals weresacrificed, and tumor volume was measured (P > 0.05). Columns, means; bars,SE. D, lung metastases at the end point of the spontaneous metastasis assaywere counted (MTLn3-B1 versus MTLn3-PL, P = 0.0001) as described inMaterials and Methods. Columns, means; bars, SE.

Figure 2. Increased ErbB1 expression enhances intravasation and tumor cellmotility. A, intravasation efficiency was determined by tumor blood burden atthe end point of the spontaneous metastasis assay. On average, 0.7 mL bloodwas directly drawn from the right ventricle and put into culture, and colonieswere counted (MTLn3-B1 versus MTLn3-PL, P = 0.0055). Columns, means of16 and 17 animals for MTLn3-B1 and MTLn3-PL, respectively; bars, SE.B, chemotaxis to EGF. Chemotaxis to EGF was tested by using a 48-wellmicrochemotaxis chamber as described in Materials and Methods. Points,means of 11 to 12 measurements from three experiments for MTLn3-B1 andMTLn3-PL, respectively; bars, SE.

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MTLn3-B1 cells show an increased chemotactic response to EGFin vitro that is consistent with the enhanced intravasation rate seenin vivo .Intravital imaging reveals that ErbB1 enhances tumor cell

motility in vivo . To determine whether increased ErbB1 expressionenhances tumor cell motility in vivo , we imaged tumor cells directlyin primary tumors in mice. MTLn3-PL and MTLn3-B1 cells werestably transduced with a GFP expression vector and then injectedinto the mammary fat pads of SCID mice to form a primary tumoras in the spontaneous metastasis assay. Four weeks after injection,animals were anesthetized, a partial skin flap dissection done toexpose the primary tumor, and the tumors were imaged using amultiphoton microscope. Tumor cell motility was identified in timelapse movies (Fig. 3A and accompanying movie) and the number ofmoving tumor cells per time lapse movie determined. There wassignificantly enhanced motility of tumor cells in MTLn3-B1 tumorscompared with MTLn3-PL tumors (Fig. 3B).To test whether the motility changes observed in MTLn3-B1

tumors reflect a difference in the tumor microenvironment ofMTLn3-B1 tumors compared with MTLn3-PL tumors, we coin-jected CFP-expressing MTLn3-B1 tumor cells with GFP-expressingMTLn3 tumor cells. On average, the same number of GFP- andCFP-labeled tumor cells were present in each imaging plane.ErbB1-overexpressing cells showed significantly greater movementand invasiveness (Fig. 3C and D), compared with neighboringnonoverexpressors. These results are consistent with increasedErbB1 expression stimulating increased tumor cell motility andinvasiveness in vivo through enhanced ErbB1 signaling and argueagainst general modifications of the tumor microenvironmentbeing the major cause of the increased motility.

This observation was confirmed in vitro using the micro-chemotaxis chamber. Equal numbers of GFP-labeled ErbB1 over-expressors were mixed with unlabeled normal expressors andexposed to 0.05 and 0.5 nmol/L EGF gradients. The relative numberof GFP-labeled to non–GFP-labeled migrating cells was found to be3 to 4 (Fig. 4A), consistent with the increased migratory capability ofErbB1 cells in response to EGF being due to an increased sensitivityafforded by the higher receptor number. The chemotactic responseto EGF of both cell lines was inhibited with dose dependenciesconsistent with the published IC50’s of 0.033 Amol/L for inhibition ofErbB1 by Iressa (ref. 34; Fig. 4B) and 0.35 Amol/L for AG825 (ref. 35;Fig. 4C), indicating that signaling from both ErbB1 and ErbB2 isrequired and consistent with the results of Fig. 1B showing thatboth ErbB1 and ErbB2 are phosphorylated in response to EGF.ErbB1-drivenmetastasis is not caused by enhanced growth of

metastases in the lungs. To determine whether increased ErbB1expression enhances lung metastasis by increasing the arrest,survival, or growth of tumor cells in the lungs, we comparedMTLn3-PL andMTLn3-B1 properties in the experimental metastasisassay. Cells were injected into the lateral tail veins of 5- to 7-week-oldfemale BALB/c SCID mice, and 2 weeks later, the mice weresacrificed, and the lungs were removed and examined for metas-tases. All animals receiving MTLn3-PL or MTLn3-B1 cells formedmetastatic lesions in the lungs. Animals injected with MTLn3-PLcells had 3.2 F 0.6 metastases/mm2, whereas animals injected withMTLn3-B1 cells had 3.3 F 0.5 metastases/mm2. There is nostatistically significant difference between the two groups (P >0.05). These results indicate that that ErbB1 overexpression inMTLn3 cells does not affect later steps of metastasis and are consis-tent with the major effect being an enhancement of intravasation.

Figure 3. MTLn3-B1 cells show higher motility in vivo. A, image showing motility of tumor cells in MTLn3-B1 tumor. GFP-expressing tumor cells (green ) andextracellular matrix fibers (purple ). White arrows track two cells present during the entire sequence; orange arrowheads show their original position. Other cellsmove into the field of view from a lower plane. Images shown are at 4- to 5-minute intervals, from a sequence in which images were taken every minute (see Movie 1).Bar, 20 Am. B, MTLn3-B1 tumor cells show increased motility and invasiveness in vivo . Average number of movement events per slice measured for MTLn3-PLand MTLn3-B1 tumors. Columns, mean for 16 slices from four tumors per cell line; bars, SE. P < 0.05, two-tailed t test. C, MTLn3-B1 CFP cells show moretranslocation than MTLn3 GFP cells. A mixture of equal numbers of MTLn3-B1 overexpressors expressing CFP and MTLn3 expressing GFP was injected into themammary fat pad, and the tumor was imaged with the multiphoton microscope using a �60 water immersion objective. GFP cells (green ) and CFP cells (purple ;for clarity). Arrowheads, three cell translocation events for the CFP cells compared with a single translocation event for GFP cells. Bar, 10 Am. D, increased MTLn3-B1cell motility is cell autonomous in vivo . The number of movement events per field for MTLn3-B1 CFP and MTLn3 GFP cells was determined and normalized forcell number by the number of CFP or GFP pixels, respectively. Columns, mean; bars, SE. On average, there were equal numbers of CFP- and GFP-labeled cellsin each field.

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Discussion

We found that increased expression of ErbB1 in MTLn3mammary adenocarcinoma cells enhanced metastasis throughsignificantly increasing intravasation. There was no effect onprimary tumor growth or the efficiency of lung colonization byinjection of tumor cells via the tail vein. In parallel withintravasation, tumor cell motility in the primary tumor in vivowas increased. This increase in motility was not due to alterationsin the tumor microenvironment, because the motility of MTLn3-B1cells was also significantly enhanced over cells expressing normallevels of ErbB1 in tumors composed of mixtures of the two celltypes. In vitro , increased expression of ErbB1 significantlyenhanced chemotaxis to EGF in a cell-autonomous fashion. Theenhanced chemotactic response was dependent on both ErbB1 andErbB2 signaling, as shown by inhibition with Iressa or AG825.To our knowledge, this is the first direct demonstration that

increased expression of ErbB1 can enhance tumor cell motility inthe primary tumor and intravasation without affecting tumorgrowth. Our studies are consistent with other in vitro andin vivo studies showing that ErbB1 expression can enhance

invasiveness (18, 19). In addition to its growth-promoting effects,ErbB1 mediates chemotaxis to EGF (36–39) and transforminggrowth factor-a (40, 41) in vitro , and increased levels of ErbB1can enhance chemotactic responses to these growth factors(39, 42–44). We have previously shown that tumor cells chemo-tax towards EGF in vivo , using an in vivo invasion assay inwhich needles containing EGF are inserted into tumors (45), andMTLn3-B1 cells show enhanced responses in this in vivo invasionassay (46). Thus, increased ErbB1 can enhance invasiveness bothin vitro and in vivo in response to ErbB1 ligands. This likelyreflects increased sensitivity provided by higher levels of ErbB1 asa heterodimer with ErbB2, which also shows increased tyrosinephosphorylation in response to EGF in MTLn3-B1 cells.Potential sources of EGF in the primary tumor that could direct

tumor cells toward blood vessels include both blood vesselsthemselves and macrophages (47–49). EGF is reported as presentin serum at around 0.1 nmol/L (50), concentrations at whichwe see a significant difference in chemotactic response betweenMTLn3-B1 and MTLn3-PL cells. Thus, gradients of EGF formedfrom leaky blood vessels could stimulate tumor cell motilitydirectly. In addition, macrophages also produce EGF and arepresent around blood vessels (1). We have shown that macrophagescan form a paracrine loop with tumor cells to enhance invasive-ness in response to gradients of EGF (46). Thus, there are multiplesources of EGF in vivo that can direct tumor cells towards bloodvessels.In summary, metastasis is an inefficient process, and it is

important to dissect each step to identify the mechanisms thatcontribute to metastasis (1, 2). A major obstacle to examiningthe intravasation step has been the lack of reliable tools to tracktumor cell movement in vivo . Critical elements required forexamination of the process of intravasation in detail include (a) amethod for evaluation of intravasation by measuring the numberof viable tumor cells present in the blood; (b) development ofmultiphoton imaging methods, which allow direct visualization oftumor behavior in vivo ; and (c) identification of tumor models,which show a strong correlation between intravasation rate andmetastatic rate. In this article, we provide an example of an appro-priate tumor model for studying intravasation efficiency and applymeasurements of intravasation and intravital imaging to showits use. These tools provide access to studies of intravasation as anew target for anticancer therapy.

Acknowledgments

Received 4/11/2005; revised 9/16/2005; accepted 10/20/2005.Grant support: National Cancer Institute grants R01 CA77522 (J.E. Segall), P01

CA100324 (J.E. Segall and J. Condeelis), and R01 CA69202 (Z-Y. Zhang).The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank the members of the Cox, Segall, and Condeelis labs for comments andsuggestions and David Stern, AstraZeneca, Bristol Myers Squibb (Princeton, NJ), andGary Nolan (Stanford University, Stanford, CA) for the reagents.

Figure 4. The increased chemotaxis of MTLn3-B1 cells in vitro is cellautonomous and depends on ErbB1 and ErbB2. A, MTLn3-B1 cells showenhanced cell-autonomous migration responses in vitro . Mixtures of 50%MTLn3-PL and 50% GFP-expressing MTLn3-B1 cells were used in 48-wellchemotaxis assays with 0.05 or 0.5 nmol/L EGF in the lower well. The relativeproportion of GFP-labeled cells to non–GFP-labeled cells migrating in responseto EGF was used to estimate the relative migration response of MTLn3-B1cells compared with MTLn3-PL cells in the same well. Columns, means of14 measurements per concentration; bars, SE. B, inhibition by Iressa of numberof cells chemotaxing in response to 0.5 nmol/L EGF in the bottom well. Dataare normalized to DMSO value. C, inhibition by AG 825 of number of cellschemotaxing in response to 0.5 nmol/L EGF in the bottom well. Data arenormalized to DMSO value.

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EGFR Enhances Motility and Intravasation In vivo

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EGFR Enhances Motility and Intravasation In vivo

In the article on how EGFR enhances motility and intravasationin vivo in the January 1, 2006 issue of Cancer Research (1), inFig. 1A , the label reading ‘‘a-ErbB4’’ should have read ‘‘a-actin.’’ Thecorrected figure appears below.

1. Xue C, Wyckoff J, Liang F, Sidani M, Violini S, Tsai K-L, Zhang Z-Y, Sahai E, CondeelisJ, Segall JE. Epidermal growth factor receptor overexpression results in increasedtumor cell motility in vivo coordinately with enhanced intravasation and metastasis.Cancer Res 2006;66:192–7.

I2006 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-66-8-COR

www.aacrjournals.org 4547 Cancer Res 2006; 66: (8). April 15, 2006

Correction