Tumor Cell Invasion Is Promoted by Interstitial - Cancer Research

Microenvironment and Immunology

Tumor Cell Invasion Is Promoted by Interstitial Flow-InducedMatrix Priming by Stromal Fibroblasts

Adrian C. Shieh1, Hallie A. Rozansky1, Boris Hinz2, and Melody A. Swartz1

AbstractInterstitial flow emanates from tumors into the microenvironment where it promotes tumor cell invasion.

Fibroblasts are key constituents of the tumor stroma that modulate the mechanical environment by matrixremodeling and contraction. Here, we explore how interstitial fluid flow affects fibroblast–tumor cell inter-actions. Using a 3-dimensional invasion assay and MDA-MB-435S cells cocultured with dermal fibroblasts in acollagen matrix, we showed a synergistic enhancement of tumor cell invasion by fibroblasts in the presence ofinterstitial flow. Interstitial flow also drove transforming growth factor (TGF)-b1 and collagenase-dependentfibroblast migration, consistent with previously describedmechanisms in which flow promotes invasion throughautologous chemotaxis and increased motility. Concurrently, migrating fibroblasts enhanced tumor cellinvasion by matrix priming via Rho-mediated contraction. We propose a model in which interstitial flowpromotes fibroblast migration through increased TGF-b1 activation and collagen degradation, positioningfibroblasts to locally reorganize collagen fibers via Rho-dependent contractility, in turn enhancing tumor cellinvasion via mechanotactic cues. This represents a novel mechanism in which interstitial flow causes fibroblast-mediated stromal remodeling that facilitates tumor invasion. Cancer Res; 71(3); 790–800. �2011 AACR.

Introduction

The tumor microenvironment contains numerous cell typesand a complex extracellular matrix (ECM), and plays a key rolein cancer progression (1, 2). Stromal fibroblasts synthesizetumor-permissive ECM, and promote tumor invasion throughsecretion of cytokines and matrix metalloproteinases (MMP;ref. 3). Less appreciated are biomechanical changes thataccompany tumor growth, such as increased matrix densityand fluid flow, yet these have significant effects (4–6). Nor-mally, fibroblasts support the epithelium by maintaining theECM, secreting cytokines, and making cell–cell contacts (7).Altered tissue mechanics, such as that caused by collagenreorganization at the tumor-stromal interface and increasedmatrix stiffness, can disrupt epithelial structures and causetumorigenesis and invasion (8–11). Because fibroblasts play a

key role in sensing mechanical stress and modulating thebiomechanical microenvironment (7, 12), we asked how thebiomechanical force of interstitial flow affected fibroblastinteractions with tumor cells.

Interstitial fluid flow through the ECM is omnipresent intissues and caused by lymphatic drainage. Because of exces-sive interstitial fluid pressure in tumors, interstitial flow andlymphatic drainage are increased in the tumor margin (13, 14).Interstitial flow can redistribute bioactive molecules and altergradients (15, 16). This in turn may drive cancer cell invasionby skewing autologously secreted chemokines, causing che-motaxis in the flow direction (17). Interstitial flow can alsoaffect stromal cells, causing cell and matrix alignment (18, 19),increasing fibroblast motility via MMP-1 (20), and inducingmyofibroblast differentiation via transforming growth factor(TGF)-b1 (18).

Because interstitial flow has significant effects on both fibro-blast and tumor cell behavior, we hypothesized that interstitialflow would have synergistic effects on tumor cell–fibroblastinteractions by altering cell behavior and the 3-dimensional (3D)matrix through which they communicate and migrate. Forexample, physical changes wrought by fibroblasts under inter-stitial flow (18) could increase matrix stiffness, density, andalignment, leading to increased tumor invasion (8, 10, 21). Weused in vitromodels that combine tumor cells and fibroblasts ina 3D environment with interstitial flow, and showed that flowdrives fibroblasts to invade in a TGF-b1– and MMP-dependentmanner, and simultaneously enhance tumor cell invasion byRho-dependent matrix priming.

Authors' Affiliations: 1Institute of Bioengineering and Swiss Institute forExperimental Cancer Research, �Ecole Polytechnique F�ed�erale de Lau-sanne (EPFL), Lausanne, Switzerland and 2Matrix Dynamics Group,Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Melody A. Swartz, SV-IBI-LLCB, Station 15,�Ecole Polytechnique F�ed�erale de Lausanne (EPFL), CH-1015 Lausanne,Switzerland. Phone: 41-21-693-9686; Fax: 41-21-693-9665. E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-10-1513

�2011 American Association for Cancer Research.

CancerResearch

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Published OnlineFirst January 18, 2011; DOI: 10.1158/0008-5472.CAN-10-1513

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Methods

Cell isolation and cultureHuman foreskin–derived dermal fibroblasts were isolated

using dispase I and Liberase Blendzyme 1 (Roche), selectedwith antifibroblast-specific antigen microbeads (Miltenyi Bio-tec) and used at passages 3 to 10. GFP-MDA-MB-435S humanmelanoma cells were used as previously described (22). MDA-MB-435S cells, IMR-90 fetal lung fibroblasts, DU-145 prostatecarcinoma, and MDA-MB-231 breast adenocarcinoma cells(ATCC) were maintained at 37�C, 5% CO2 in Dulbecco'smodified Eagle's medium (DMEM) with L-glutamine andsodium pyruvate (PAA Laboratories), 10% fetal bovine serum(FBS), and antibiotics (Invitrogen).

3D invasion assayModified 3D Boyden chambers, as previously described (17),

were used to measure tumor cell and fibroblast invasion.Tumor cells (5 � 105 cells/mL) and/or fibroblasts (2.5 �105 cells/mL) were suspended in 1.8 mg/mL rat tail tendontype I collagen and 0.5 mg/mL Matrigel (BD Biosciences). Insome cases, Matrigel was omitted. For fibroblast densityexperiments, between 5 � 105 and 50 � 105 fibroblasts/mLwere used. The cell–ECMmixture was added to culture insertswith 8-mm pores (Millipore). After gelation, DMEM with 5%FBS (serum-free for TGF-b1–blocking experiments andserum-free with 0.1% bovine serum albumin for exogenousTGF-b1 experiments) was added. A pressure head of 5 mmH2O drove tumor-relevant interstitial flow of approximately0.5 mm/s (13). As appropriate, 0.45 mg/mL heparan sulfate,0.45 mg/mL chondroitin sulfate A, 0.3 U/mL heparinase III,100 mmol/L blebbistatin, 10 mmol/L ML-7 (Sigma-Aldrich), 1mg/mL mouse anti-human TGF-b1 antibody or mouse IgG1

isotype control (R&D Systems), rhTGF-b1 (PeproTech), 10mmol/L GM6001 (Millipore), and 2 mg/mL cell-permeableC3 transferase (Cytoskeleton Inc.) were added to the matrixand medium. Fibroblasts were labeled with 1 mmol/L Cell-Tracker Green CMFDA or 2 mmol/L CellTracker Red CMPTX(Invitrogen).

TGF-b1 measurementTotal TGF-b1 was measured using the Human TGF-b1

DuoSet ELISA (R&D Systems). Active TGF-b1 was measuredusing transformed mink lung cells (TMLCs) that produceluciferase under the plasminogen activator inhibitor-1 pro-moter (23). TMLCs (2.5 � 106 cells/mL) were cocultured withtumor cells and/or fibroblasts under static or flow conditions.Cell lysate was incubated with luciferin (Promega) and lumi-nescence was measured on a Centro LB luminometer (Bert-hold Technologies).

Dye-quenched collagen proteolysis assayA total of 2.5 � 105 fibroblasts/mL were suspended in the

previously described collagen/Matrigel ECM with 100 mg/mLDQ collagen type I (Invitrogen) and seeded into a modifiedradial flow chamber (19). Cells were exposed to static or flowconditions for 18 hours and then imaged using an LSM 510META confocal microscope (Carl Zeiss AG). The fluorescence

intensity was quantified around each fibroblast using ImageJafter subtracting background fluorescence.

Gelatin zymographyMedium samples from the 3D invasion assay were resolved

on a 10% gelatin/SDS-PAGE gel (Bio-Rad) under denaturing,nonreducing conditions. The MMPs were renatured, and thegels were incubated overnight at 37�C in developing buffer toinduce proteolysis. Gels were stained with 0.5% Coomassie R-250 and imaged with a ChemiDoc XRS (Bio-Rad).

Confocal and live-cell microscopyFor live-cell microscopy experiments, cell-seeded radial flow

chambers were placed on the confocal microscope in a 37�C,5% CO2 chamber for time-lapse fluorescence and reflectanceimaging under static or flow conditions. Images were takenevery 10 to 15 minutes for 18 hours. Afterwards, 2% parafor-maldehydewas flowed through the chambers for later imaging.

Gel contraction assayAfter a 3D invasion assay, the cell-seeded gels were removed

(initial diameter 10 mm) and incubated in culture medium.After 6 hours, the contracted diameter of the gels was mea-sured to calculate the change in area.

Data and statistical analysesAll data are presented as mean � SEM. For 3D invasion

assay results, cell counts were normalized to the matchingcontrols from each experiment, as noted in each figure legend.

Statistical analyses were done using Matlab R2008 (Math-Works); t tests and multifactor analysis of variance (ANOVA)were used. For experiments that used multiple batches of cellsand lots of matrix, an additional blocking variable wasincluded to account for these variations. If the ANOVA showedsignificance at P < 0.05, a Tukey's Honest Significant Differ-ences test was used for post hoc multiple comparisons.

Results

Interstitial flow stimulates fibroblast and concomitanttumor cell invasion

We first verified that MDA-MB-435S tumor cells, culturedfor 18 hours alone in a 3D collagen/Matrigel matrix, were moreinvasive under a physiologic level of approximately 0.5 mm/sinterstitial flow (Fig. 1A) than in static conditions, a phenom-enon we previously identified in 3D Matrigel culture andcoined "autologous chemotaxis" (17). Without Matrigel, whichis rich in heparan sulfate and binds many chemokines andgrowth factors (24), tumor cells did not respond to interstitialflow (Fig. 1A). Because in our earlier study, we found thatautologous chemotaxis was dependent on the chemokineCCL21 (17), we hypothesized that matrix binding of CCL21via heparan sulfate may play an important role in autocrinegradient formation and amplification (15). Consistent withthis hypothesis, we found that soluble heparan sulfate, but notchondroitin sulfate, significantly decreased interstitial flow-mediated tumor cell invasion (Supplementary Fig. 1A),presumably by competitively inhibiting CCL21 binding to

Flow-Induced Fibroblast Invasion Promotes Tumor Invasion

www.aacrjournals.org Cancer Res; 71(3) February 1, 2011 791




matrix-associated heparan sulfate for subsequent pericellulargradient formation. Treatment with heparinase III, whichselectively cleaves heparan sulfate and thus prevents CCL21binding to matrix heparan sulfate, also decreased interstitialflow-mediated tumor cell invasion (Supplementary Fig. 1B).Thus, interstitial flow stimulated tumor cell invasion througha 3D collagen/Matrigel matrix in a heparan sulfate-dependentmanner, and we hypothesize that this was due to its chemo-kine binding activity that is required for local gradientsto form.

Next, we examined the effects of cocultured fibroblasts ontumor cell invasion.We found that in static conditions, 2.5� 105

fibroblasts/mL cocultured with MDA-MB-435S cells resulted ina small increase in tumor cell invasion (Fig. 1B); however, under0.5 mm/s interstitial flow, they markedly increased tumor cellinvasion compared with tumor cells cultured alone underinterstitial flow (Fig. 1B). Note that the response elicited by

combining fibroblasts and interstitial flow was significantlygreater than the summative effects of either fibroblasts or flow.Similar effects were observed using IMR-90 fibroblasts (Supple-mentary Fig. 2) and MDA-MB-231 and DU-145 cell lines (Sup-plementary Fig. 3). This fibroblast-enhanced tumor cell invasionwas not due to soluble factors, because the effect was abolishedwhen fibroblasts were cultured in an adjacent gel upstream oftumor cells (Fig. 1B). Increased tumor cell invasion correlated toincreased fibroblastmigration, as interstitial flow also increasedfibroblastmigration, and evenmore so in the presence of tumorcells (Fig. 1B). Thus, we hypothesized that increased fibroblastmigration and physical interactions between tumor cells andfibroblasts were necessary for fibroblasts to enhance tumor cellinvasion under flow.

Unlike tumor cells, flow-enhanced fibroblast invasion wasindependent of Matrigel (Fig. 1C). As a result, tumor cellscontinued to invade in the presence of fibroblasts and flow,

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Figure 1. Fibroblasts enhance tumor cell invasion in the presence of interstitial flow. Normalized cell invasion for MDA-MB-435S tumor cells (TC) andhuman dermal fibroblasts (Fb). In all graphs, invasion was normalized to the leftmost condition except in D, in which invasion was normalized to the lowestfibroblast density in static conditions. A, tumor cell invasion (without fibroblasts) in the presence or absence of 0.5 mm/s interstitial flow through a 1.8 mg/mLcollagenmatrix containing either 0.5 mg/mLMatrigel (MG) or noMatrigel. B, tumor cell (left) or fibroblast (right) invasion in coculture in the presence or absenceof flow. Left, the cocultured fibroblasts were either mixed throughout the matrix with the tumor cells or present in a contiguous, but separate matrix upstreamof the tumor cells. C, effects of Matrigel on flow-enhanced tumor cell (left) or fibroblast (right) invasion. D, effects of cocultured fibroblast density on flow-enhanced invasion of tumor cells. *, P < 0.05 versus matching static condition; #, P < 0.05 between indicated groups (A–C) or versus lowest fibroblastdensity (D).

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Cancer Res; 71(3) February 1, 2011 Cancer Research792




even when Matrigel was absent (Fig. 1C); this is in contrast totumor cells alone, which did not respond to flow withoutMatrigel (and its heparan sulfate proteoglycans; Fig. 1A). Thissuggests that fibroblasts enhanced tumor cell invasion via amechanism independent of tumor cell autologous chemotaxis(17). Finally, fibroblast- and flow-enhanced tumor cell inva-sion was dependent on fibroblast density being greater than50,000 cells/mL (Fig. 1D). Fibroblast density had no effect ontumor cell invasion present in static conditions, consistentwith the hypothesis that increased tumor cell invasion due tofibroblasts and flow was dependent on fibroblast migration.

Flow- and fibroblast-enhanced tumor cell invasiondepends on TGF-b1Because previous work has shown that interstitial flow can

stimulate fibroblast TGF-b1 expression (18) and mechanicalstress can activate latent TGF-b1 (25), we hypothesized thatflow-enhanced fibroblast invasion involved TGF-b1 activation.Whenwe blockedTGF-b1 activitywith a neutralizing antibody,the fibroblast-enhanced response of tumor cells to interstitialflow was abrogated (Fig. 2A). TGF-b1 blocking did not affecttumor cell invasion in the absence of flow or fibroblasts, butonly when both were present (Fig. 2A). Similarly, flow-enhanced fibroblast migration was also inhibited by TGF-b1blocking (Fig. 2A). These results suggest that TGF-b1 wasindirectly enhancing tumor cell invasion by directly affectingfibroblast migration in the presence of interstitial flow.To determine how TGF-b1 affected cell migration, we

quantified the concentration and activity of TGF-b1. TotalTGF-b1 did not change between static and flow conditions,and was resident in the matrix and produced by fibroblasts(Fig. 2B). Using reporter TMLCs that produced luciferase inresponse to active TGF-b1, we observed increased TGF-b1activity in response to interstitial flow, regardless of the celltypes present (Fig. 2C). The lower TGF-b1 activation seen ingels containing fibroblasts (Fig. 2C) compared with TMLCsalone was likely due to competition between the 2 cell typesfor active TGF-b1, because fibroblasts consume TGF-b1. Sig-nificantly, increased TGF-b1 activation occurred even whenreporter TMLCs alone were present (Fig. 2C), suggesting eitherthat interstitial flow can directly activate latent TGF-b1(which is present in serum and Matrigel), independent oftumor cells and fibroblasts, or that interstitial flow increasedTGF-b1 availability through improved mass transport.To determine whether increased TGF-b1 was directly indu-

cing tumor cell invasion, we exposed tumor cells to exogenousTGF-b1, either uniformly (Fig. 2D, left) or in a gradient(Fig. 2D, center). In both cases, tumor cell invasion was eitherinsensitive to or inhibited by exogenous TGF-b1. Combinedwith the lack of an effect when neutralizing TGF-b1 in tumorcells alone (Fig. 2A), this suggested that TGF-b1 was actingprimarily on the fibroblasts. Treating fibroblasts with TGF-b1,or tumor cells cocultured with fibroblasts, did not stimulatefibroblast or tumor cell invasion (Fig. 2D, right). ExogenousTGF-b1 combined with interstitial flow elicited a bimodalfibroblast invasion response, with peaks at 0 and 2 ng/mLTGF-b1, and a similar pattern of tumor cell invasion in thepresence of fibroblasts (Fig. 2D, right). However, over the

range of concentrations tested, fibroblast and tumor cellinvasion never exceeded control values, and, in general,TGF-b1 inhibited fibroblast and tumor cell invasion. Biphasicmigration responses have been previously reported in bothfibroblasts and endothelial cells (26, 27), but these resultsshowed peak migration at an intermediate, optimal concen-tration of TGF-b1. Given that fibroblasts invaded more underflow than in the presence of TGF-b1 (with or without flow),but neutralizing TGF-b1 completely abolished flow- andfibroblast-enhanced tumor cell invasion (Fig. 2A), we hypothe-sized that localized, active TGF-b1 gradients, rather than anincrease in TGF-b1 concentration, may drive flow-enhancedfibroblast migration. This increase in fibroblast migration, inturn, is correlated with enhanced tumor cell invasion.

Fibroblast- and flow-enhanced tumor cell invasiondepends on MMPs

MMPs (secreted by fibroblasts and other cells) are oftenimplicated in tumor invasion (28). We found that the broadspectrum MMP inhibitor GM6001 significantly decreasedtumor cell invasion when both interstitial flow and fibroblastswere present, but had no effects in the absence of eitherfibroblasts or interstitial flow (Fig. 3A). GM6001 also decreasedflow-induced fibroblast invasion (Fig. 3A). Previous studieshave shown that interstitial flow increases fibroblast motilitythrough upregulation of MMP-1 (20), so we next used DQcollagen to determine whether collagenolysis was altered withflow. Indeed, fibroblasts exposed to interstitial flow exhibited a70% increase in pericellular collagen degradation, and col-lagen degradation tended to coincide with increased matrixconsolidation around fibroblasts (Figs. 3B and C). However,gelatin zymography revealed minimal levels of active MMP-2/9, which are often implicated in tumor-stromal interactions(29), and no differences in total MMP-2 or MMP-9 activitybetween static and flow conditions were found, or betweentumor cells cultured alone versus with fibroblasts (Fig. 3D).This is consistent with previous reports that MMP-1, and notMMP-2, was responsible for interstitial flow-mediatedenhancement of fibroblast motility (20). Thus, interstitial flownot only increased fibroblast invasion via TGF-b1, but alsothrough increased collagen degradation.

Rho-dependent fibroblast contractility drives flow-enhanced tumor cell invasion

Cell contraction is an important mechanism by whichfibroblasts alter their surroundings (30). ECM contraction isalso important in tumor invasion, because stromal stiffnesscorrelates with tumorigenesis and invasion (8–10), and theability to deform the ECM is instrumental for tumor cell andfibroblast invasion (11, 21, 31). We inhibited fibroblast con-traction pathways by targeting Rho activation with C3 trans-ferase, myosin light chain kinase (MLCK) with ML-7, andnonmuscle myosin IIA with blebbistatin. Rho and MLCK areupstream effectors of fibroblast contraction (32, 33), whereasmyosin IIA is the downstream cog in actomyosin interactions(34). We observed significant differences in fibroblast mor-phology on treatment with C3 transferase and blebbistatincompared with ML-7 (Fig. 4A). C3- and blebbistatin-treated






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Figure 2. TGF-b1 is necessary for flow-enhanced tumor cell invasion only when fibroblasts are present. Invasion results are normalized to the leftmostcondition in each graph unless otherwise noted. A, effects of TGF-b1 neutralization on MDA-MB-435S (TC) invasion (left) or fibroblast (Fb) migration (right)using a function-blocking antibody (a-TGF-b1 block). B, total TGF-b1 levels asmeasured by ELISA after 18 hours culture with 5� 105 tumor cells/mL and 2.5�105 fibroblasts/mL. C, active TGF-b1 levels as measured by TMLC luciferase reporter assay, after 18 hours culture with 2.5 � 106 reporter cells/mL.The first condition contains only TMLC reporter cells; all other conditions contain the cells noted plus TMLCs. D, left, effects of exogenous TGF-b1 on theinvasion of tumor cells alone. Middle, effects of an exogenous TGF-b1 gradient on the invasion of tumor cells alone. Right, effects of exogenous TGF-b1 ontumor cell invasion (in the presence of fibroblasts) and fibroblast invasion alone. Tumor cell and fibroblast invasion are normalized to their respective flow,no TGF-b1 condition. *, P < 0.05 versus matching static condition; #, P < 0.05 between indicated groups.

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fibroblasts showed significantly altered cell morphology,reduced collagen matrix contraction, and few stress fibers(Fig. 4A). In contrast, ML-7–treated fibroblasts exhibited nor-mal morphology and still contracted the matrix (Fig. 4A).The morphologic differences induced by the different inhi-

bitors matched the tumor cell invasion and fibroblast migra-tion results. When we specifically inhibited Rho activation infibroblasts with a cell-permeable C3 transferase, the flowresponse of tumor cells was impaired (Fig. 4B). This effectwas independent of fibroblast migration, as C3 treatment

actually increased fibroblast migration (Fig. 4B). This wasconsistent with previous findings showing that Rho/Rho-kinase inhibition can actually increase cell migration (33,35). Similarly, blebbistatin treatment specifically inhibitedonly flow- and fibroblast-enhanced tumor cell invasion(Fig. 4C). Like C3 treatment, blebbistatin-treated fibroblastsexhibited increased migration (Fig. 4C). Conversely, ML-7decreased tumor cell and fibroblast invasion under all con-ditions (Fig. 4D), showing that MLCK was not specificallynecessary for flow- and fibroblast-enhanced tumor cell

Figure 3. MMP activity isnecessary for flow- and fibroblast-enhanced tumor cell migration. A,effects of MMP inhibition onMDA-MB-435S tumor cell (TC) invasion(left) or fibroblast (Fb) migration(right). Invasion is normalized tothe leftmost condition in eachgraph. B, confocal fluorescenceimages of fibroblasts (red, F-actin)and degraded collagen [green,fluorescein isothiocyanate (FITC)].Scale bar, 25 mm. C, quantificationof FITC intensity (normalized tothe static condition) for DQcollagen experiments. D, gelatinzymogram on supernatantsfrom 3D cultures. *, P < 0.05versus matching static condition;#, P < 0.05 between indicatedgroups.

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invasion. These findings show that Rho-mediated fibroblastcontractility is essential to fibroblast- and flow-enhancedtumor cell invasion.

Fibroblasts mediate ECM reorganizationTo determine whether fibroblast contractility was affected

by flow, we used confocal reflectance microscopy to observethe collagen fiber matrix around fibroblasts and tumor cells.We observed fibroblasts contracting ECM fibers in theirimmediate vicinity in both static and flow conditions(Fig. 5). Fibers aligned with spindle-shaped fibroblasts or withcell processes when fibroblasts adopted a dendritic morphol-ogy. Flow itself did not affect ECM contraction by fibroblasts,as determined both microscopically (Fig. 5) and with a col-

lagen gel contraction assay (Supplementary Fig. 4). This showsthat two factors-–increased fibroblast migration in responseto interstitial flow and the fibroblast's innate propensityfor cell contraction-–must interact to enhance tumor cellinvasion.

In many instances, tumor cells extended processes towardfibroblasts, and in other cases tumor cells and fibroblastsmade direct contact (Fig. 5). Tumor cell processes generallyextended along the direction of fibroblast-aligned matrixfibers. In live-cell imaging experiments, tumor cells understatic and flow conditions showed limited capacity to deformthe matrix (Supplementary Movies 1 and 2). When fibroblastswere present, we observed significant ECM displacement,likely due to fibroblast contraction (Supplementary Movie

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Figure 4. Flow- and fibroblast-enhanced tumor cell migration depends on Rho-mediated fibroblast contractility. Invasion results are normalized to theleftmost condition in each graph unless otherwise noted. A, representative confocal images of fibroblasts seeded in 1.8 mg/mL collagen gels containing0.5 mg/mL Matrigel. Green, F-actin; red, collagen matrix (confocal reflectance). Denser matrix indicates collagen fiber consolidation and contraction.Scale bar, 30 mm. B, left, effects of C3 transferase treatment (C3) specifically in fibroblasts (Fb) on invasion of MDA-MB-435S cells (TC). Right, effects of C3treatment on fibroblast invasion. C, effects of blebbistatin treatment (blebbi) on tumor cell invasion (left) or fibroblast migration (right). D, effects of ML-7treatment on tumor cell invasion (left) or fibroblast migration (right). *, P < 0.05 versus matching static condition; #, P < 0.05 between indicated groups.

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3). When both fibroblasts and flow were present, the ECMshowed significant displacement (Supplementary Movie 4).Notably, the matrix appears more degraded than in otherconditions, suggesting that increased proteolysis, as we andothers have previously shown (20), is involved in the invasionand remodeling process. We also observed tumor cells inter-acting with and aligning alongside fibroblasts (SupplementaryMovie 4). The changes and interactions in SupplementaryMovie 4 were representative. We conclude that fibroblastslocally remodeled the collagen matrix by applying tractionforces to their surroundings, causing local changes in matrixdensity and fiber alignment, and in turn facilitating interac-tions with tumor cells.

Discussion

The importance of the tumor microenvironment on cancergrowth and invasion are well appreciated (1–3), and thestiffness of the tumor stroma has been shown to drivetumorigenesis and invasion (8–10). However, in addition tomatrix stiffness, interstitial flow is an important mechanicalstress in the tumor stroma (5). By examining the interplaybetween tumor cells, fibroblasts, and interstitial flow, weshowed that flow guides fibroblast invasion, leading to con-current invasion of MDA-MB-435S tumor cells through theECM.Without interstitial flow, fibroblasts did not affect tumorcell invasion.TGF-b1 regulates a variety of tumor suppressive and

promoting effects, including epithelial homeostasis, epithe-lial-to-mesenchymal transition, myofibroblast differentia-

tion, and metastasis (36). TGF-b1 was necessary forinterstitial flow-enhanced fibroblast invasion (Fig. 2A) butonly indirectly involved in tumor cell invasion (Fig. 2D). Wehypothesize that TGF-b1 may increase fibroblast invasionthrough both TGF-b1–driven autologous chemotaxis (17, 37)and increased cell motility (38). Because exogenous TGF-b1did not enhance (and in some cases inhibited) tumor celland fibroblast invasion, this suggests that flow-generatedgradients of TGF-b1, which would be washed out by exo-genous TGF-b1, may be crucial. We also determined thatinterstitial flow leads to an apparent increase in TGF-b1activation (Fig. 2C). This may be due to the direct effect ofinterstitial flow, consistent with other findings that fibro-blast contraction and fluid flow can activate TGF-b1 (25, 39),which typically involves proteases and integrins (40, 41).Although the shear stress generated by the levels of inter-stitial flow examined here are likely extremely low (42),matrix fiber organization can strongly increase local shearstresses on cells and their pericellular matrix (43), poten-tially leading to mechanical activation of TGF-b1. Interstitialflow may also increase the availability of TGF-b1 by facil-itating transport of active growth factor, which would like-wise lead to an apparent increase in TGF-b1 activity. Thus, itis likely that the flow-enhanced fibroblast invasion wascaused by increased TGF-b1 activation or improved trans-port, leading to increased fibroblast motility and/or chemo-taxis.

Collagen degradation was also necessary for flow-inducedfibroblast invasion and concurrent tumor cell invasion(Fig. 3A). Consistent with previous work with adventitial

TC + Fb static

TC + Fb flow

flow direction flow directionflow direction flow direction

TC only static

TC only flow

Figure 5. Fibroblasts locally contract the matrix and interact with tumor cells. Confocal fluorescence/reflectance images of MDA-MB-435S cells (TC; green),fibroblasts (Fb; red), and collagen matrix (white) under static and flow conditions. In many instances, tumor cells extend processes toward fibroblasts,in the direction of contracted collagen fibers, or align with fibroblasts (arrowheads). Scale bar, 25 mm.






fibroblasts, myofibroblasts, and smooth muscle cells (20), wefound increased pericellular collagen degradation by fibro-blasts in response to flow (Fig. 3B). Although collagen pro-teolysis may be important primarily for cell motility (20), itmay also facilitate ECM remodeling, as has been previouslyshown with fibrosarcoma and breast carcinoma cells (44).

Our findings suggest that, similar to prior work with othertumor cell types (31, 45), fibroblasts may locally remodel thematrix via Rho-dependent mechanisms as they migratethrough the ECM, priming the matrix such that invadingtumor cells invade much more rapidly. Fibroblasts controlECM remodeling (7, 12) partly by exerting traction on the ECMvia Rho-dependent contractility (30, 32). When fibroblastscontract, fibers align with their pseudopodia and stress fibers(30). Previous studies have shown that Rho and MLCK playnonredundant roles in regulating myosin activation; Rho andRho kinase are involved in central stress fiber formation andcell contraction, whereas MLCK affects stress fibers at the cellperiphery and cell motility (32, 33, 46, 47). Inhibition of Rhoand myosin IIA actually increases cell motility (33–35); this islikely due to the fact that Rho promotes stress fiber formationfor traction force generation, whereas migration may requiremore selective adhesion and less force exertion. These resultscorroborate our findings that Rho, but not MLCK, was neces-sary for fibroblast matrix contraction and subsequent matrixpriming (Fig. 4B-D). Rho and nonmuscle myosin IIA inhibitionincreased fibroblast invasion even while simultaneouslydecreasing fibroblast- and flow-enhanced tumor cell invasion(Fig. 4B and C). These data also suggest that fibroblastmigration is necessary, but not sufficient, to enhance tumorcell invasion. Fibroblasts must also remodel the matrixthrough Rho-dependent cell contraction; thus, if fibroblastsare migrating more but contracting less as they move (asshown in Fig. 4), they would be unable to enhance tumor cellmigration.

On the basis of our findings, we propose a mechanism inwhich interstitial flow drives TGF-b1– and MMP-dependentfibroblast invasion, during which fibroblasts remodel theECM through Rho-mediated cell contractility to prime themicroenvironment for tumor cell invasion (Fig. 6). Fibro-blasts secrete latent TGF-b1; fibroblast contraction andinterstitial flow may enhance TGF-b1 activation, and inter-stitial flow can also increase the availability of TGF-b1.When TGF-b1 is activated pericellularly and nonuniformlyredistributed due to interstitial flow, this drives fibroblastchemotaxis in the flow direction. Interstitial flow alsostimulates fibroblast collagenolysis, leading to increasedcell motility (20). Simultaneously, tumor cells invade thematrix by CCR7-dependent autologous chemotaxis (17);thus, both fibroblasts and tumor cells are invading con-currently but independently. As the fibroblasts invade, theyalso apply traction forces to the collagen matrix, creatinglocal lines of tension resulting in alignment of collagenfibers and increased matrix density near the fibroblast(Fig. 5). Previous work has shown that active fibroblastcontraction, combined with the nonlinear properties ofECM and local anisotropy, creates mechanical gradients,stiffens the matrix both locally and globally, and transmits

forces between cells up to a distance of 500 mm (48). This issupported by time-lapse confocal images (SupplementaryMovies 3 and 4), in which fibroblasts generate matrixdeformation and prime the microenvironment for tumorcell invasion, which depends on collagen alignment, matrixdensity, and stiffness (11, 21, 31, 49, 50). Importantly, we didnot observe increased fibroblast contraction by interstitialflow; however, fibroblasts are already sufficiently contractileunder static conditions to meaningfully remodel the ECM(Fig. 5; Supplementary Movies 3 and 4). Instead, the keyevent in the proposed model is stimulation of increasedfibroblast invasion by interstitial flow. When fibroblastsmigrate more, they effectively increase their range of influ-ence, encountering more tumor cells and coincidentallypriming the local matrix to encourage tumor invasion.

In summary, we present a novel mechanism wherebythe biomechanical force of interstitial flow enhances fibro-blast invasion and leads to the priming of the matrix micro-environment, making it more permissive for tumorcell invasion. Interstitial flow acts as a biomechanical"switch," inducing fibroblasts to prime the stroma for tumorinvasion. Thus, interstitial flow can drive the invasive behaviorof tumor cells, not only through autologous chemotaxis (17)but also by changing the nature of tumor–fibroblast interac-tions. These findings expand our current view of the tumormicroenvironment, which not only should encompass cells,soluble factors, and the ECM, but also must include

latent TGF-β1binds to the matrix

activeTGF-β1

fibroblast

tumorcell

interstitial flow increasesactivation and/or

availability of TGF-β1

fibroblasts prime matrix by Rho-

mediated contraction

tumor cell invades usingprimed matrix

TGF-β1 andcollagen degradationenhance fibroblast

migration

interstitial flow interstitial flowstimulates

MMP activity

degradedcollagen

Figure 6. Hypothesized mechanism of flow- and fibroblast-enhancedtumor cell migration. Stromal fibroblasts secrete latent TGF-b1 that bindsECM. Interstitial flow enhances activation and availability of TGF-b1,stimulates collagen degradation, and increases fibroblast migration. Asthe fibroblast moves, it primes the ECM through Rho-dependentcontractility. Nearby tumor cells (already invading by CCR7-dependentautologous chemotaxis) take advantage of the primed matrix to enhancetheir invasion.

Shieh et al.





biomechanical forces like interstitial flow and how theseforces interact with the tumor and the other facets of themicroenvironment.

Disclosure of Potential Conflicts of Interest

The authors have no conflicts of interest to disclose.

Acknowledgments

The authors thank Jacqueline Shields, Jan Overney, Ulrike Haessler, AmineIssa, Pierre-Jean Wipff, and Martin Seneviratne for assistance and advice, DanielRifkin for providing the TMLCs, andMihaela Skobe for providing the GFP-MDA-MB-435S cells.

Grant Support

The work has received funding from by the Whitaker International Fellowsand Scholars Program (to A.C. Shieh), Susan G. Komen for the Cure (KG080585to M.A. Swartz and A.C. Shieh), Oncosuisse (02114-08-2007 to M.A. Swartz), theNational Centre for Competence in Research in Molecular Oncology, theCanadian Institutes of Health (#488342 to B. Hinz), and the EU FP7 InternationalTraining Network 3TNet (to B. Hinz).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 3, 2010; revised October 29, 2010; accepted December 3, 2010;published OnlineFirst January 18, 2011.

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2011;71:790-800. Published OnlineFirst January 18, 2011.Cancer Res Adrian C. Shieh, Hallie A. Rozansky, Boris Hinz, et al. Matrix Priming by Stromal FibroblastsTumor Cell Invasion Is Promoted by Interstitial Flow-Induced

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