NHE1 promotes invadopodial ECM proteolysis through acidification of the peri-invadopodial space

The FASEB Journal • Research Communication

NHE1 promotes invadopodial ECM proteolysis throughacidification of the peri-invadopodial space

Giovanni Busco,*,1 Rosa A. Cardone,*,1 Maria R. Greco,* Antonia Bellizzi,‡

Matilde Colella,* Ester Antelmi,* Maria T. Mancini,* Maria E. Dell’Aquila,†

Valeria Casavola,* Angelo Paradiso,‡ and Stephan J. Reshkin*,2

*Department of General and Environmental Physiology and †Department of Animal Production,University of Bari, Bari, Italy; and ‡Clinical Experimental Oncology Laboratory, National CancerInstitute Giovanni Paolo II, Bari, Italy

ABSTRACT Extracellular matrix (ECM) degradationis a critical process in tumor cell invasion and requiresmembrane and released proteases focalized at mem-brane structures called invadopodia. While extracellu-lar acidification is important in driving tumor invasion,the structure/function mechanisms underlying this reg-ulation are still unknown. Invadopodia are similar instructure and function to osteoclast podosomes respon-sible for bone degradation, and extracellular acidifica-tion is central to podosome action, suggesting that itcould also be for invadopodial function. Here, utilizinga novel system for in situ zymography in native matrices,we show that the Na�/H� exchanger (NHE1) andNHE1-generated extracellular acidification are local-ized at and necessary for invadopodial-dependent ECMdegradation, thereby promoting tumor invasion. Stim-ulation with EGF increased both NHE1-dependent pro-ton secretion and ECM degradation. Manipulation ofthe NHE1 expression by RNA interference or activityvia either transport-deficient mutation or the specificinhibitor cariporide confirmed that NHE1 expressionand activity are required for invadopodia-mediatedECM degradation. Taken together, our data show aconcordance among NHE1 localization, the generationof a well-defined acidic extracellular pH in the nano-space surrounding invadopodia, and matrix-degradingactivity at invadopodia of human malignant breastcarcinoma cells, providing a structural basis for the roleof NHE1 in invasion and identifying NHE1 as a strate-gic target for therapeutic intervention.—Busco, G.,Cardone, R. A., Greco, M. R., Bellizzi, A., Colella, M.,Antelmi, E., Mancini, M. T., Dell’Aquila, M. E.,Casavola, V., Paradiso, A., Reshkin, S. J. NHE1 pro-motes invadopodial ECM proteolysis through acidifica-tion of the peri-invadopodial space. FASEB J. 24,3903–3915 (2010). www.fasebj.org

Key Words: metastasis � pHe � MDA-MB-231 � breast cancer

Tumor invasion and metastasis associated with neo-plastic progression are the major causes of cancerdeaths. The invasive process occurs through a complexseries of interactions with the host tissue resulting in

the proteolysis, penetration, and infiltration of thenormal tissue by the cancer cell (1). Although it is wellknown that misregulation of tissue remodeling path-ways contributes to a variety of pathologies includingcancer (2, 3), where invasion in solid tumors is associ-ated with altered receptor activity and increased expres-sion and activity of secreted extracellular proteases (4,5), the mechanisms that regulate these processes arestill poorly known.

Recent advances have highlighted the importance ofthe acid component of the tumor microenvironment indriving tumor invasive capacity (6–8) and subsequentmalignant progression (9, 10). This can occur via thedirect destruction of the surrounding normal tissueand extracellular matrix (ECM; refs. 11–14) or throughtheir enzyme-directed proteolysis via the extracellularpH (pHe)-dependent up-regulation of protease secre-tion/activation (6, 7, 10, 14–21). Further, there isincreasing evidence that the activity of the NHE1, aregulator of intracellular pH, is essential for invasion invarious tumor cell types (22, 23) and that its activity isalso known to play a role in the acidification of thetumor microenvironment (19) and to be necessary forprotease activation (7, 19, 21, 22, 24). However, thecellular structural basis for these functions is still un-known.

The discovery of invadopodia as discrete ECM-de-grading structures on the surface of invasive and met-astatic tumor cells has provided an important newinsight into the cellular and molecular basis of tumorinvasion. Invadopodia are dynamic protrusive struc-tures in invasive cancer cells that are associated withECM proteolysis and increased invasive capacity, and ahallmark of invasive tumor cells is the degradation ofthe ECM by invadopodia through proteases and pro-tease-associated molecules enriched in invadopodia(25–27). Based on all these data, we hypothesized thatNHE1 may also be involved in focal proteolytic activity

1 These authors contributed equally to this work.2 Correspondence: Department of General and Environ-

mental Physiology, University of Bari, Via Amendola 165/A,70126 Bari, Italy. E-mail: [email protected]

doi: 10.1096/fj.09-149518

39030892-6638/10/0024-3903 © FASEB

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at these sites of cell invasion through its acidification ofthe peri-invadopodial space.

Here, we demonstrate a concordance amongNHE1 localization, extracellular acidification, andproteolytic, matrix-degrading activity in invadopodiaof human malignant breast carcinoma cells. Indeed,we observe that nanospace extracellular acidification,as in osteoclastic bone remodeling, plays a funda-mental part in the invadopodial-dependent proteol-ysis of the ECM by breast cancer cells. In the tumorcells, this acidification is driven by the activity of theNHE1 localized to the invadopodia together withactin and cortactin. Further, utilizing the techniquedeveloped in this study, we were able to extend ourobservations to frozen breast cancer biopsies and weobserved that NHE1 was colocalized to proteolyticmembrane structures extending from the tumor.Thus we propose that the NHE1 acidifies the peri-invadopodial space and, in turn, this acidificationup-regulates invadopodial morphology and proteo-lytic activity. These findings provide, for the firsttime, a structural basis for the well-known role of theNHE1 in tumor cell invasion and its regulation ofprotease activity and identify NHE1 as a strategictarget for anti-invasion therapeutic intervention.

MATERIALS AND METHODS

Cells and expression vectors containing NHE1 mutants

MDA-MB-231 human breast carcinoma cells were grown inDMEM high glucose supplemented with 10% of FBS and1% Pen/Strep and maintained in a 5% CO2 incubator. Forlow pH experiments, cells were grown in complete highglucose DMEM in which bicarbonate was added at concen-trations calculated to produce the desired pH in the 5%CO2 incubator.

GFP-NHE1 (pCMV6 plasmid) was purchased fromGeneCopoeia (Rockville, MD, USA), and pCMV plasmidcontaining the ion transport dead E226I-NHE1 mutant waskindly supplied by Prof. Diane Barber (University of Cali-fornia, San Francisco, CA, USA). Plasmid cDNA or emptyvector was incubated with Trans-IT-LT1 transfection re-agent (Mirus, Dharmacon Lafayette, CO, USA) in a ratio of3:1 (3 �l reagent/�g DNA) as described by the companyprotocol. Incubation was performed for 24 h. Cells seededon matrices were then treated, and focal/pericellular ECMdigestion or invadopodia shape was measured.

Reagents

Gelatin from porcine skin, type A was purchased fromSigma (St. Louis, MO, USA). Matrigel growth factor re-duced without phenol red was from BD Bioscience (SanJose, CA, USA). DQ-Green-BSA, DQ-Red-BSA, and DQ-pigskin gelatin fluorescein conjugated were from MolecularProbes (Eugene, OR, USA). Primary antibodies used forWestern blot were as follows: monoclonal [4E9] anti-NHE1(Abcam, Cambridge, MA, USA), monoclonal anti-cortactin(p80/85) clone 4F1 (Millipore, Beford, MA, USA), andpolyclonal anti-MMP2 (Cell Signaling, Danvers, MA, USA).Rhodamine B, isothiocyanate, and mixed isomers (tetra-methylrhodamine isothiocyanate) were purchased from

Sigma. Polyclonal anti-NHE1 (Alpha Diagnostic, San Anto-nio, TX, USA), monoclonal anti-cortactin (p80/85) clone4F1 (Millipore), and monoclonal anti-HA-tag 262K (CellSignaling) were used for immunofluorescence. Secondaryantibodes were anti-mouse (Sigma) and anti-rabbit (CellSignaling) horseradish perioxidase-linked antibodies, goatanti-mouse and anti-rabbit Alexa Fluor 568 linked, anddonkey anti-mouse Alexa Fluor 647 linked (MolecularProbes).

NHE1 RNA interference

MDA-MB-231 cells were transfected with 100 nM smallinterfering (si)RNA targeting NHE1 (NHE1 siRNA) or 100nM nonspecific siRNA as control (scrambled) using poolsof 4 siRNA duplexes each designed to target human NHE1(siGenome SMART pool M-005277-00-0005, humanSLC9A1, NM_003047, Dharmacon, Lafayette, CO, USA) asdescribed previously (28). NHE1 activity was analyzed 72 hpost-transfection with either NHE1 siRNA or control,scrambled siRNA and was found to be inhibited also by�60% (data not shown). Two days after transfection withthe indicated siRNA oligos, cells were plated on Matrigel,and 24 h later, the amounts of pericellular and focaldigestion were measured as described below.

ECM digestion using in situ zymography

In vitro dequenching assay

Experiments were conducted in metastatic breast cancer cellsseeded onto a layer of Matrigel (diluted to a final concenta-zion of 4 mg/ml) in which quenched BODIPY linked to BSA(DQ-Green-BSA and DQ-Red-BSA) was mixed at a finalconcentration of 30 �g/ml. The matrix mix was used to cover12-mm round glass coverslip. Matrigel containing the fluori-genic substrate was allowed to polymerize for 30 min in ahumidified incubator at 37°C. Then, 30,000 cells/coverslipwere seeded onto the polymerized matrix and grown for 6, 8,and 24 h. Before fixation, proteolysis activity could be moni-tored under the cell body by using an inverted epifluores-cence microscope. After the above-mentioned times, cellswere fixed with paraformaldehyde 3.7% in PBS and processedfor immunofluorescence. Invadopodia-dependent ECM di-gestion was evaluated microscopically. Focal proteolysis pro-duces fluorescence within a black background, which is usedboth to quantitatively measure proteolytic activity levels andin colocalization analysis.

In vivo clinical tissue slice system

The distribution of NHE1 and its associated metastatic signal-ing proteins (cortactin) in relation to ECM proteolysis andpHe in human preneoplastic and invasive epithelial lesionswas analyzed by 3-D immunomicroscopy. In vivo ECM prote-olysis was assayed by layering collagen containing DQ-gelatin-FITC on top of the tumor slices and measuring the produc-tion of fluorescence as extracellular proteases digest it asdescribed above.

Cross-linked gelatin layer preparation and fractionation

Porcine skin gelatin was dissolved to a final concentration of2 mg/ml in PBS containing 2 mg/ml of sucrose by slightlywarming in a microwave oven. Fifteen milliliters of gelatinsolution, kept warm at 40°C, was spread on 150-mm-diameterplastic dishes to evenly cover the entire dish surface. Excess

3904 Vol. 24 October 2010 BUSCO ET AL.The FASEB Journal � www.fasebj.org

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gelatin was removed, and the layer that remained was allowedto gel on ice for 10 min. Then, 10 ml of ice-cold, 0.5%glutaraldhyde in PBS was added to each dish to cross-link thegelatin on ice for 15 min with gentle shaking. The cross-linked gelatin was then washed 3 times with PBS for 5 min,and 20 ml of 70% ethanol was added to each dish for 1 hunder a sterile bench hood to sterilize. The gelatin layer waswashed 2 times with sterile PBS for 5 min and 2 times withcomplete DMEM; the last wash of DMEM was not removedand the dishes were left in a humified, 37°C incubator for 1 hfollowed by seeding 4 � 106 cells on each dish. After 24 h ina humified, 37°C, 5% CO2 incubator, cell fractions wereisolated as follows: each dish was washed 3 times with PBScontaining 1 mM CaCl2 and 0.5 mM MgCl2 and 2 times with0.2� PBS plus 1 mM CaCl2 and 0.5 mM MgCl2. Cells werethen incubated on ice with 3 ml of hypertonic swelling buffer[0.2� PBS supplied with 2 �l/ml protease inhibitor cocktail(Sigma), 1 mM PMSF, and 1 mM sodium orthovanadate] for15 min on ice. Cell bodies were gently scraped with anL-shaped pasteur and centrifuged at 10,300 g for 30 min.Supernatant was collected (cytosolic soluble proteins) andplaced on ice while the pellet was resuspended with 100 �l oflysis buffer (5 mM HEPES, 0.5 mM EDTA pH7.2 suppliedwith 2 �l/ml protease inhibitor, 1 mM PMSF, 1 mM sodiumorthovanadate, 1 mM DTT, and 0.1% Nonidet), and mem-brane proteins were extracted by 30 min of rotating at 4°C.After 2 washes with PBS, the entire gelatin layer containingentrapped invadopodia was scraped from the dish with 1 mlof above lysis buffer. The collected gelatin was vortexed, andprotein was extracted for 30 min on an orbiting wheel at 4°C.The fractions containing membrane proteins or invadopodiawere collected in Eppendorf tubes and centrifuged at 13,000rpm at 4°C. The supernatant of each fraction was collected,and the pellet was discarded. Protein concentration of the 3fractions were measured with Bradford (Pierce, Rockford, IL,USA). The diluted samples were precipitated overnight with 9vol of acetone at �20°C and centrifugated at 10,300 g for 1 h.Proteins were resuspended in SDS sample buffer (6.25 mMTris-HCl, pH 6.8, containing 10% glycerol, 3 mM SDS, 1%2-mercaptoethanol, and 0.75 mM of bromphenol blue) con-taining DTT.

pHe determinations and measurement of NHE1 activity

The pH of areas of extracellular proteolysis was measured byfluorescence ratio imaging of unquenched fluorescine-conju-gated gelatin (DQ-FITC-gelatin; Molecular Probes) that hadbeen liberated by proteolysis of the ECM in which it wasdissolved. In brief, an aliquot of Matrigel (ice-cold) wasdiluted to 4 mg/ml with ice-cold serum-free DMEM andDQ-FITC-gelatin (1 mg/ml) was added at a final concentra-tion of 30 �g/ml. This substrate was used to coat the glassbottom of sterile WillCo-dish confocal culture dishes (WillCoWells BV, Amsterdam, Netherlands), and the dishes were leftin a 37°C, humidified incubator for �30 min for solidifica-tion. Then, 105 cells/dish were seeded 12 h before theexperiment and returned to the incubator. The disheswere then mounted, and areas of digested ECM in eachfield were imaged on a Nikon Eclipse TE 2000S epifluores-cence microscope (Nikon Instruments SpA, SestoFiorentino, Italy) equipped with a MicroMax 512BFT CCDcamera (Princeton Instruments, Princeton, NJ, USA) usinga Nikon lamp shutter with a mercury short arc photo opticHBO 103 W/2 lamp for excitation (Osram, Augsburg,Germany) and an �40 Nikon Sfluor oil objective. Imageswere acquired with 490-nm (pH sensitive) and 440-nm(isosbestic pH insensitive) excitation wavelength filters,using a 535-nm emission filter, and analyses were per-formed with the MetaFluor 4.6r9 software (Universal Im-

aging Corp., Marlow, UK). The use of the ratio of apH-sensitive and -insensitive signals permits an analysis ofpH that is continously corrected for any changes in probeconcentration that could occur during the experiment.Experiments were conducted as follows: digested areaswere selected and after signal stabilization (to determinethe steady state ratio value), an extracellular alkalinizationwas produced by perfusing the cell layer with a NaCl Ringer(140 mM NaCl, 5 mM KCl, 20 mM HEPES, 10 mM glucose,0.1 mM CaCl2, and 1 mM MgCl2) at pH 7.4 in which thefluid level of the dish was maintained by needle aspiration,and pHe was followed over time. After a lag time thatvaried between 4 and 6 min, cells began to reacidify theareas of digested ECM and the initial rate of Na�-dependent acidification was determined by linear regres-sion analysis of the first 15 points taken at 4 s intervals afterthe re-equilibrium of the focal or pericellular zones atpHe 7.4. After the extracellular area had reacidified, theentire process was repeated to determine its reproducibil-ity. The activity of the NHE1 was measured by monitoringthe rate of the second pHe reacidification in the presenceof NaCl Ringer (pH 7.4) containing 500 nM cariporide(HOE642) to block NHE1 activity. This concentration isabout twice the measured IC50 for cariporide inhibition ofthe NHE1 in these cells (see Fig. 5B) when measuring pHiwith BCECF-AM after an intracellular acidification with theammonium chloride prepulse (29). After each experiment,in situ calibration curves were obtained by perfusing thecells in the dish with a NaCl ringers having different pH(6.2, 6.8, 7.2, 7.4, and 7.8). At the end of the experiment,the dish was incubated with a ringer containing 0.5 mg/mlof crystal violet to quench all external fluorescence (30).The experimental data were exported to an Excel sheet, abackground ratio was subtracted from each ratio value ineach analyzed area, and the calibration ratio values wereused to build a calibration curve (ratio-pH) to convert eachratio value into a pH value. From each analyzed area, thefollowing information was obtained: 1) the initial, steadystate, beginning pHe value; 2) the velocity (slope ofreacidification/minute) with which the cell is able toreacidify the areas of ECM digestion after a forced alkalin-ization; and 3) the extracellular acidification activity of theNHE1.

Coimmunofluorescence

Cells were seeded on a layer of gelatin (2 mg/ml) or Matrigel(4 mg/ml) containing a substrate for digestion localization(DQ-FITC-gelatin, BSA-BODIPY-FL, or RED-DQ-BSA at afinal concentration of 30 �g/ml), and coimmunofluores-cence was performed as described previously (28).

Image acquisition and analysis

Images were acquired and analyzed as described previously(28, 31) with an �60 oil objective using a Nikon Eclipse TE2000S epifluorescence microscope or a laser scanningconfocal microscope (C1/TE2000-U; Nikon Instruments).Confocal images were analyzed using ImageJ (http://rsb.info.nih.gov/ij/), and membrane colocalization wasdetermined by analyzing the whole acquired images usingthe bidimensional correlation function JACoP. The ran-dom or codependent nature of the above-calculated appar-ent dye-overlap colocalizations were tested using intensitycorrelation analysis (ICA) and intensity correlation quo-tient (ICQ), as described previously (31). ICA plots canidentify regions with dependent, random, or segregatedstaining relationships, and the ICQ value represents a

3905STRUCTURAL BASIS FOR NHE1-DRIVEN TUMOR INVASION

single quantitative value for each region of interest wherepositive values increasing to a maximum of 0.5 indicateever greater significant positive colocalization, values �0indicated random colocalization, and negative values in-creasing to a maximum of �0.5 indicate ever greatersignificant segregated localization. RGB profiles, whichdemonstrate the intensity fluorescence distribution foreach fluorophore, were obtained using the ImageJ plugin.

RESULTS

NHE1 colocalizes with ECM degradation in vitro andin vivo

While NHE1 is known to be involved in regulatingcancer cell invasion, pseudopodial protrusion, proteaseactivation, integrin receptor function, and motility, itsstructural localization during invasion and role in ECMdegradation is yet to be determined (22, 23). As astarting point of the study of the pH regulation of focalinvadopodial-dependent ECM degradation, we mea-sured the association of NHE1 expression with focalproteolytic ECM degradation and with the expressionof the actin nucleation factor and invadopodia markercortactin. We first utilized the classic technique inwhich cells are plated on top of a chemically cross-linked layer of gelatin labeled with rhodamine andfocal digestion is observed as the removal of back-ground red substrate fluoresence. As shown in Fig. 1,cells of the highly invasive human breast cancer lineMDA-MB-231, incubated on this gelatin for 6 h,removed focal zones of gelatin observed as nonfluo-rescent areas both under the cell body and at the cellperiphery. Confocal immunofluorescence analysis ofthe expression of the invadopodial marker, cortactin

(blue), and either endogenous NHE1 (Fig. 1A,green) or exogenous GFP-NHE1 (Supplemental Fig.1A) showed that these two proteins were associatedwith areas of focal digestion and, further, were oftencolocalized in various focal digestive zones.

As has been observed for other proteins, such as Arf6and DRFs (32, 33), the expression of neither protein islimited to the invadopodia but is also heavily expressedin other cellular districts and this generalized expres-sion can render their observation in small structuressuch as invadopodia somewhat difficult. To bettervisualize invadopodia entrapped in the ECM, we re-moved the cell body of cells incubated for 24 h on 2%cross-linked porcine gelatin following a modified Muel-ler protocol (34) and then either extracted the proteinfrom the different cellular fractions and performedWestern blotting for cortactin, MMP2, focal adhesionkinase (FAK), and NHE1 (Fig. 1B); or directly per-formed confocal immunofluorescence for cortactinand NHE1 expression in the invadopodia that re-mained in the gelatin (Fig. 1C) or exogenous GFP-NHE1 (Supplemental Fig. 1B). Figure 1B shows thatNHE1 and cortactin were both found in the invadopo-dial fraction, while FAK, as has been recently reported(35), was not found in the invadopodia. In confocalimmunofluorescence inside the gelatin layer, NHE1and cortactin were observed to colocalize in variousgroups of invadopodia (Supplemental Fig. 1B). Statis-tical analysis of their colocalization by cytofluorogramand by ICA of the variance in the distribution of the 2proteins (28, 31) demonstrated that cortactin andNHE1 localization at invadopodia was highly codepen-dent (Supplemental Fig. 1C; ICQ�0.327�0.035; n�7;P�0.001) and, in both NHE1-labeling approaches, Z

Figure 1. NHE1 is expressed in invadopodia. Invadopodia protein expression on cross-linked gelatin layers. A) Confocalimmunofluorescence micrograph of MDA-MB-231 cells cultured on cross-linked rhodamine (tetramethylrhodamine isothio-cyanate)-conjugated gelatin. Matrix degradation is visualized as loss of red fluorescence background, and cells were stained withanti-cortactin antibody (blue) and anti-NHE1 antibody (green). There were widespread, focalized zones of degradation, andcortactin and NHE1 were both localized and often colocalized in digested areas, as shown in the 2 representative sagittal (XZ)sections (arrowheads). B, C) To better visualize invadopodia entrapped in the ECM, the cell bodies of cells incubated for 24 hon a thick layer of cross-linked gelatin were removed and separated into cytosol and plasma membrane, while entrappedinvadopodia were extracted from the gelatin as described in Materials and Methods. B) Proteins of the 3 fractions were assayedin Western blot analysis. C) With the use of the above protocol on coverslips, immunofluorescence was directly performed forNHE1 (green) and cortactin (red) in the invadopodia that remained in the cross-linked gelatin. Representitive immunofluo-rescence of 4 independent experiments. Axial (XY), sagittal (XZ), and coronal (YZ) sections show a colocalization of NHE1 andcortactin in invadopodia. Scale bars � 10 �m (A); 5 �m (C).


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analysis showed that the invadopodia were �1 �m inwidth and ranged from 3 to 5 �m in length.

This traditional technique is limited by the thinnessof the ECM layer, by its extreme rigidness and resis-tance to digestion derived from the chemical cross-linking, and by possible alterations in cell-matrix inter-actions and availability of released nutrients. Indeed, ithas been suggested that the invadopodial structurecould be an artifact of the cross-linked 2-D nature of thein vitro system used and that differences in matrixconditions could induce substantial changes in theinvadopodial structure or dynamics/behavior (27, 36)and even in invasive/motile behavior (14). Further-more, the localization of invadopodial activity by theremoval of entrapped fluorescence in the ECM doesnot permit a quantitative analysis of proteolytic activity.Therefore, we developed a modified protocol based onthe degradation-dependent release of fluorescence of aquenched fluorophore (i.e., DQ-Green-BSA) that hasbeen dissolved in a thicker, non-cross-linked matrixlayer (37–39) comprised either of a single ECM com-ponent such as collagen I or of the natural, complexmixture found in Matrigel (40). In this protocol, diges-tion is observed as the appearance of unquenchedfluorescence in a dark background rather than as theremoval of high background fluorescence. Importantly,this protocol also gives a more sensitive measure of theinitiation and/or low levels of digestion and, further,permits the quantification of local proteolytic activity,the high-resolution mapping of individual focal cleav-age sites and a more exact confocal colocalizationbetween focal digestion and proteins of interest. Pre-liminary experiments in cells cultured on DQ-Green-BSA-Matrigel for 6 h demonstrated that, indeed, eithercortactin (Supplemental Fig. 2A) or NHE1 (Supple-

mental Fig. 2B) was colocalized in the areas of observedfocal proteolysis of ECM bound DQ-Green-BSA in ahighly significant manner in Matrigel.

When cultured on DQ-Green-BSA-Matrigel for24 h and examined in 3-color confocal fluorescencemicroscopy, MDA-MB-231 cells formed small punc-tate actin/cortactin rich structures highly associatedwith focal ECM digestion that were detected asbrilliant fluorescent green spots (Fig. 2A). Z section-ing revealed that these structures protruded verti-cally from the ventral membrane into the Matrigelsubstrate at sites of focal ECM degradation. RGBanalysis of either the XY planar view (not shown) orZ sections showed that the indeed actin/cortactincolocalization occurred in areas of focal digestionand with a size of the proteolytic structures (inva-dopodia) very similar to those observed above oncrossed-linked gelatin: �1 �m in diameter and 3–5�m in length. ICA/ICQ analysis of the Z sectionsdemonstrated that this colocalization was significant(ICQ�0.386�0.015; n�14; P�0.001). These proper-ties of the actin/cortactin punctate digestive struc-tures are clearly consistent with that of invadopodiareported previously (41– 45) and “invadopodia-like” structures formed in Matrigel by the same cellline (33).

Therefore, we next examined the correlation be-tween NHE1/cortactin expression and ECM focal di-gestion in 3-color fluorescence confocal microscopy incells cultured on DQ-Green-BSA-Matrigel for 24 h. Inthese experiments, NHE1 also highly colocalized withboth cortactin and focal ECM proteolysis both alongthe edge of the cell as well as at the tip of leading edgepseudopodia (Fig. 2B). The sizes of these NHE1-ex-pressing proteolytic, invadopodial structures were very

Figure 2. NHE1 is expressed in invadopodiaof cells on Matrigel. To better visualize inva-dopodial focal digestion and protein localiza-tion in Matrigel, we developed an in situzymography technique using the quenchedfluorescent substrate DQ-Green-BSA, such

that a quantifiable fluorescence is released only on digestion of the matrix. Cells seeded on Matrigel were allowed to digestthe fluorigenic substrate, and after fixation cells were assayed in immunofluorescence. A, B) Images show cortactin andactin (A) or NHE1 and cortactin immunolocalization (B) at BSA proteolytic spots (green) in axial planes taken at thebottom of the cells (XY). XY-YZ-zoomed sections of representative regions of interest, indicated by numbers 1–3 in eachfield, are shown at bottom. Letters a–c indicate individual invadopodia in the respective region of interest 1. As can be seenin green, low levels of basal, diffuse digestion were observed under and around the cells together with more restricted areasof focal digestion. Representitive immunofluorescence of 7 independent experiments. C) Immunofluorescence micro-graph of NHE1 (red) and proteolysis (green) in frozen sections from a human lobular breast carcinoma tissue.Representitive immunofluorescence of 3 independent experiments. Left panel: low-magnification XY plane of the section,displaying a series of lobules. Center panel: magnification of the boxed lobule from the left panel. Right panels: YZ planeand relative magnification of indicated area show the high colocalization of NHE1 with proteolysis. Scale bars � 10 �m (A,B); 100 �m (C, left panel); 50 �m (C, center panel).


similar to those observed above in Fig. 2A. As has beenreported for colocalization of cofilin and N-WASP (46)or of cortactin and MT1-MMP (47), the images shownin both Figs. 1A and 2B suggest that NHE1 andcortactin could have different expression dynamics thatalter their relative presence in the various stages ofinvadopodia formation and activity.

It has been further suggested that the invadopodialstructure might not only be an artifact of the in vitroculture systems in which they are studied but, fur-ther, that this structure may not exist in in situhuman tumors (25–27, 48). Up to now, in vivodetection of ECM proteolysis in clinical human can-cer tissues has been limited to the analysis of the levelof degradation at low magnification due to the lackof reliable techniques to visualize invadopodia in atumor and to the inherent limits of the traditionaltechnique of visualizing proteolysis via the removalof entrapped fluorescence (38). Here, we applied theabove fluorescence release-based technique for thein situ localization of protease activity in frozen tissuesections from a breast cancer patient (38, 49). In Fig.2C, an XY planar view of a 5-�m tissue section of apT3N1aM0G3 breast cancer biopsy is presented inwhich focal ECM digestion (DQ-Green-gelatin) andNHE1 (red) can be observed colocalized (yellow) instructures that, when analyzed in Z section, arereminiscent in shape of invadopodia observed in thein vitro measurements. ICA/ICQ analysis demon-strated that this colocalization was significant(ICQ�0.185�0.011; n�5; P�0.01).

Altogether, these studies demonstrate that invasivebreast cancer cells form invadopodia in various in vitroand in vivo ECM substrate systems and that NHE1 is,indeed, localized in invadopodia.

NHE1-dependent hot spots of acidic pHe areassociated with focal ECM digestion and areaugmented by EGF

Fundamental questions are whether areas of invadopo-dial focal ECM proteolysis are also acidic and whetherthe NHE1 has a role in driving acidification in theseareas. As a starting point of the study of the pHregulation of focal invadopodial-dependent ECM deg-radation, MDA-MB-231 cells were plated on Matrigelcontaining gelatin labeled with quenched fluorescein(FITC), DQ-FITC-gelatin. After 24 h of proteolysis ofthe ECM and consequent release of the dequenchedfluorescence, the cells produced areas of strong focal,invadopodial fluorescence (Fig. 3A, right panel, arrow-heads) and a weak generalized proteolysis underneaththe entire cell that we are calling pericellular prote-olysis. The pHe of digested Matrigel at zones of focaland pericellular ECM digestion was determined byutilizing the pH sensitivity of the FITC (30, 50)released by proteolysis of the quenched DQ-FITC-gelatin (see Materials and Methods; SupplementalFig. 3). The ratiometric method using the ratio of apH insensitive and pH-sensitive excitation wave-lengths of FITC permits an analysis of pH that is

Figure 3. Peri-invadopodial space has an acidic pHe, and NHE1 is responsible for this acidification. To examine levels of pHeand the mechanisms regulating pHe in the above areas of focal ECM digestion in Matrigel, we developed a technique based onthe pH sensitivity of released FITC from quenched DQ-FITC-gelatin dissolved in the Matrigel. A) Typical microscopy fields. Leftimage: MDA-MB-231 in brightfield; right image: associated diffuse and focal proteolytic spots (white puncta in blackbackground, as indicated by red arrowheads). B) Steady-state acidic pHe measured in a single region of interest of active focalproteolysis and the reacidification observed after 2 forced alkalinizations by perfusion with Ringer at pH 7.4. C) Sameexperiment in which the second reacidification was performed in the presence of the NHE1 inhibitor cariporide (HOE642),which inhibited reacidification in the focal proteolytic area. D) Rate of reacidification plotted against the intial, steady-stateextracellular pHe created around invadopodia. The acidic pHe recovery rate after forced extracellular alkalinization is directlyproportioned to the intial pHe value (n�27). E) Immunofluorescence images showing that stimulation with 100 ng/ml EGFincreased focal proteolytic activity in Matrigel containing DQ-Green-BSA. F) Typical trace of a pHe experiment showing that 100ng/ml EGF increased the rate of reacidification recovery after alkalinization in focal proteolytic areas. Scale bars � 20 �m (A);10 �m (E). Asterisks indicate end of latency period.


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continously corrected for changes in probe concen-tration. Figure 3A displays typical fields in transmit-ted light (left panel) and FITC fluorescence showingcells and areas of digestion (right panel), respec-tively. Areas of focal proteolysis had a mean pHevalue significantly lower than areas next to cellswhere only pericellular digestion had occurred[mean pHe 6.36�0. 37 (range 6.2–7.2) vs. 7.43�0.15(range 7.35–7.51) for the focal and pericellular,respectively; n�40; P�0.001; Supplemental Fig. 4].This relatively high value of pericellular pHe at theedge of the cell is consistent with that recentlyreported for motile melanoma cells on collagen I(51), and the larger range of very low pHe values inzones of focal digestion may be due to variability insize and activity of the invadopodia. Figure 3 displaystypical traces of reacidification after perfusion withRinger pH 7.4 and followed by a second round ofalkalinization in the absence (Fig. 3B) or presence(Fig. 3C) of 500 nM of the specific NHE1 inhibitorcariporide (HOE642). When the cells were perfusedwith Ringer at pH 7.4, the areas of extracellular focaldigestion rapidly alkalinized and then, after a latencyperiod of 5.3 � 1.2 min (n�25), began to reacidify.As can be observed, the second round of alkaliniza-tion produced a slightly higher acidification rate ofthe focal areas of digestion than the first round(0.031�0.007 vs. 0.034�0.008 pHe U/min for firstand second trace, respectively; n�23; P�0.05) andcariporide blocked this reacidification by 76.14 �2.06% (0.029�0.011 vs. 0.0063�0.0012 pHe U/minfor minus and plus cariporide, respectively; n�20;P�0.001), supporting the functional role of NHE1 inthis process. Further, there was a strong associationbetween the rate of reacidification and initial steady-state pHe values when the rate of extracellular acid-ification for each focal area of digestion is plottedagainst its initial pHe value (Fig. 3D).

EGF signaling is known to finely regulate NHE1activity (52) and to drive both chemotaxis/invasion(52) and invadopodial formation/function (46, 53).Figure 3E shows that EGF treatment did, indeed,increase focal ECM proteolysis in our cells. To deter-mine if EGF stimulation preferentially acidifies thefocal areas of ECM digestion, cells seeded on DQ-FITC-gelatin-Matrigel were allowed to form areas offocal invadopodial digestion for 24 h. Figure 3Fdisplays typical reacidification traces after perfusionwith Ringer, pH 7.4, and followed by a second roundof alkalinization after 10 min preincubation of 100ng/ml EGF (rate of acidification 0.026�0.003 vs.0.038�0.0041 pH U/min for control and EGF-stimulated cells, respectively; n�12; P�0.001). Theobserved acidification of the focal zones of ECMproteolysis and the further strong, rapid increase inacidification during EGF stimulation are consistentwith an increased capacity for net acid extrusion. Incontrast, EGF had no effect on total cellular expres-sion of NHE1 in these conditions (Supplemental Fig.5), demonstrating that it is, indeed, changes in its

localized activity and not expression that drives itsregulatory control of invasive activity in these meta-static breast cells.

Extracellular acidification augments invadopodialECM degradation by increasing invadopodial numberand length

These data raise the questions of whether this low-ered focal pHe has a role in driving focal ECMdegradation and, if so, is it through an increase ininvadopodial number and/or activity. Experimentalextracellular acidification has been shown to stronglyincrease osteoclast podosome proteolytic activity byactivating osteoclastic proteolytic programs (54, 55).In analogy to these studies, we next examinedwhether exposure to a similar experimental acidosiscould increase focal ECM degradation in MDA-MB-231 cells plated on Matrigel containing DQ-Green-BSA-BODIPY and whether this increase occurred viaa direct increase in invadopodial activity and/ormorphology. As can be seen in Fig. 4A, B, extracel-lular acidification (from pHe 7.4 to 6.8) increasedthe level of focal ECM digestion, measured as thepixel density of focal zones of digestion for each cell,while having no significant effect on the levels ofpericellular digestion of the ECM, measured as thepixel density per cell exclusive of the focal areas.Confocal images of typical cells from the pHe 7.4(Fig. 4C) and the pHe 6.8 (Fig. 4D) treatments,respectively, show that exposure to extracellular acid-ification produced a significant increase in both totalinvadopodia expression and length observed in thecell. We conclude that, as in osteoclasts, an experi-mental acidification increases tumor cell proteolyticactivity by turning on intrinsic invadopodial pro-grams and that an important future direction will beto identify the molecular components of these spe-cific invasive programs.

NHE1 expression and transport activity regulatesinvadopodial-dependent ECM degradation andinvadopodia formation

To further address the question of whether NHE1 isinvolved in the regulation of tumor cell focal proteolyticactivity, we analyzed the effect of knocking down itsexpression or specifically inhibiting its transport activityon ECM proteolytic ability and invadopodia structure inMDA-MB-231 cells. Cells were plated onto Matrigel con-taining DQ-Green-BSA-BODIPY, and the effect of thefollowing treatments on both the pericellular and focalproteolytic activity of the cell was measured: 1) transienttransfection of 100 nM of an siRNA against NHE1;2) dose-response treatment with the specific NHE1 inhibitorcariporide; or 3) transient transfection of a HA-tagged iontransport-defective mutant of NHE1 (E266I; refs. 56, 57).

Figure 5A shows that 72 h of siRNA treatmentresulted in an �60% reduction of NHE1 expressionmeasured in immunofluorescence (left panel) or West-


ern blot (middle panel), comparable to that previouslyreported (28). The analysis of Matrigel proteolysis (Fig.5A, right panel) measured by the release of BODIPYfluorescence revealed that siRNA treatment also re-duced focal proteolysis by �60%, while pericellularproteolysis was unaltered. Cariporide treatment re-duced both focal digestion (Fig. 5B, left and centerpanels) and NHE1 activity (Fig. 5B, right panel) withsimilar dose-response relationships of cariporide con-centration on focal ECM digestion (center panel) orNHE1 activity (right panel) (IC50 of 180�21 vs. 205�15nM for NHE1 and focal digestion, respectively; n�25;not significant). The same results were obtained forboth siRNA and cariporide treatment in cells plated oncross-linked Oregon Green 488-gelatin in which diges-tion removes fluorescence (data not shown). Lastly,cells transfected with the ion transport-defective mu-tant of NHE1 (E266I, HA-positive cells) had a greatlyreduced amount of focal Matrigel digestion comparedwith nontransfected cells (Fig. 5C, left panel). Analysisof a large number of cells demonstrated that, indeed,cells transfected with the E266I mutant of NHE1 hadlevels of focal digestion that were comparable to thelevels of diffuse pericellular proteolysis (Fig. 5C, rightpanel).

The effects of siRNA treatment on cortactin contain-ing invasive structures or the E266I mutation on HA-NHE1 containing invasive structures were next mea-sured in confocal microscopy. As can be seen in Fig. 6A,cells treated with scrambled RNA had well-formedinvadopodia with cortactin strongly associated withfocal digestion (left panel), while in an siRNA-treatedcell cortactin was highly expressed in large numbers ofsmall clusters above the ventral surface of the cell (right

panel). In E266I-transfected cells, HA-NHE1 expres-sion, as above for cortactin in siRNA treated cells, wasobserved to be limited to small intracellular clustersabove the ventral cell surface (Fig. 6B).

DISCUSSION

To invade into tissues and cross tissue boundaries,aggressive cancer cells produce specialized, highly dy-namic adhesive structures, termed invadopodia, thatdrive matrix degradation by focal proteolytic activity vialocalized proteases. Because extracellular acidificationand the NHE1 are involved in tumor protease cascades(see Introduction), we hypothesized that, in analogy toosteoclast podosomal-dependent bone proteolysis, anNHE1-dependent, highly acidic extracellular environ-ment is formed in the nanospace surrounding theinvadopodia and that this is essential for efficientproteolytic activity. Our structural and molecular/bio-chemical findings support this hypothesis and demon-strate that invadopodia are acidification structuresdriven by the activity of NHE1 localized to their mem-brane. Furthermore, our experiments in native ECMand in frozen clinical histological sections extend thedata beyond previously utilized protocols and provideevidence that focal acidification and digestion of theECM are important components of both basic andEGF-stimulated tumor invasion.

We observed that NHE1 is localized to invadopodiautilizing both the classic cross-linked, single compo-nent fluorescence disappearance technique (Fig. 1)and a new, fluorescence appearance based protocolin Matrigel (Fig. 2) developed specifically for this

Figure 4. Low extracellular pH increases invadopodialmatrix proteolysis. To examine the effect of pHe oninvadopodial density and size and proteolytic ability ofthe cells, MDA-MB-231 cells were seeded onto Matrigelcontaining DQ-Green-BSA on coverslips in completegrowth medium for 2 h in order to attach and spread.Medium was then changed to fresh complete growthmedium at either pH 7.4 or 6.8, and the cells were left todigest the ECM for an additional 24 h. Amount ofdigested Matrigel was assayed as described in Materialsand Methods. A) Typical fields of actin (red)-stainedcells and proteolysis (green) in the 2 pHe conditions. Ascan be seen, low levels of basal, pericellular digestion

were observed under and around the cells together with more restricted areas of focal digestion. B) Total amount of bothpericellular (peri) and focal digestion measured in pixel density, as described in Materials and Methods. Data are means �se (n�16). *P � 0.01, ***P � 0.001 vs. cells incubated at pHe 7.4. C, D) Confocal images of a typical cell incubated at pHe7.4 (C) or 6.8 (D) show cortactin (red) and digestion (green) colocalization in protrusive digestive structures on the ventralcell surface. Scale bars � 10 �m (A, C, D); 5 �m (C, D insets).


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study. We also demonstrate that, in analogy to osteo-clast podosomal-dependent bone proteolysis, anNHE1-dependent, highly acidic extracellular envi-ronment is formed in the nanospace surrounding theinvadopodia (Fig. 3) and, in turn, this acidificationup-regulates invadopodia morphology and proteo-lytic activity (Fig. 4). Through the siRNA-drivenknockdown of NHE1 expression or the blockage ofits activity with either the selective NHE1 inhibitorcariporide or the transport negative E266I mutation,we observed that the activity of NHE1 is essential forinvadopodia-dependent focal ECM digestion (Fig. 5)and invadopodia formation (Fig. 6). As selectiveinhibition of the NHE1 also abrogates invasion (22),we conclude that NHE1 orchestrates invasion, at leastin part, through its regulation of invadopodial-dependent focal proteolytic activity. We further vali-dated this thesis by correlating the level of NHE1expression in immunofluorescence with the amountof focal ECM degradation for a series of MDA-MB-231 cells, and, indeed, as shown in Fig. 7, the level ofNHE1 expression in each cell was highly correlatedwith its digestive capacity. Altogether, these data

strongly suggest that regulation of ECM degradationclosely follows the regulation of NHE1 activity, sup-porting the fundamental role that NHE1 plays intumor cell invasion. Further, these data tie togetherthe large series of observations demonstrating thatNHE1-dependent activity, via the acidification of theextracellular space, functions together with extracel-lular proteases to digest the ECM in a controlledmanner and drive invasion (7, 19, 21, 22, 24). Animportant future research direction will be dedicatedto elucidating the proteases present at or surround-ing the invadopodia and the dynamics of both theirregulation by extracellular pH and their role indriving ECM proteolysis and in regulating invadopo-dial function through, yet unknown, protease-activated receptors and/or release of entrapped fac-tors.

There is a current debate as to the relative impor-tance of protease-dependent cleaving of ECM fibrils vs.protease-independent amoeboid-like behavior in driv-ing tumor cell motility through 3-D ECM (for a recentreview on the 2 types, see ref. 58). Recently, it has beensuggested these two choices might depend on the

Figure 5. NHE1 is necessary for invadopodiaproteolytic activity. To better understand therole of NHE1 in invadopodial-dependent fo-cal digestion of the ECM, MDA-MB-231 cellswere transfected with siRNA against NHE1(A), treated with increasing concentrationsof the specific NHE1 inhibitor cariporide(HOE642; B), or transfected with a transportdead mutant of NHE1, E266I (C). A) Cellstransfected with a pool of siRNA specific forNHE1 were analyzed for NHE1 expression byeither immunofluorescence (left panel) orWestern blot analysis with anti-NHE1 anti-body (middle panel) 3 d after transfection.The smart pool of siRNA against NHE1mRNA decreased NHE1 expression and focalproteolysis to similar extents while having noeffect on pericellular digestion. Right panel:expression of NHE1 in immunofluorence andthe total amount of both pericellular (greenbars) and focal (blue bars) digestion ex-pressed in pixel density. Data are means � se(n � 16). †††P � 0.001 for NHE1 expression;***P � 0.001 for focal proteolysis. B) Leftpanel: typical fields showing actin expression(red) and ECM proteolysis (green) in cellstreated with vehicle or 1 �M cariporide, re-spectively. Center and right panels: dose-re-sponse curves for the effect of cariporidetreatment on relative NHE1 activity and focalECM digestion, respectively. NHE1 activity,measured by BCECF-AM spectrofluorimetry,and pHi recovery were assayed, and the initialrate of the NHE1 activity was calculated asNa�-dependent H� efflux. All measurements

were conducted in nominally HCO3�-free, HEPES-buffered solutions in order to measure only the NHE1 activity. C) Effect of

transport dead mutant, E266I, on ECM digestion. Left panel: typical field showing anti-HA recognition of the transfectedexogenous NHE1 expression, ECM digestion, and merge of the 2 fields. Right panel: quantification of pericellular and focalproteolytic ability is expressed as pixel density of HA negative and positive cells, respectively. Graph shows that transfected,E266I-expressing cells maintained their pericellular digestive ability, while their ability to focal digest was dramatically decreasedwith respect to the control, untransfected cells. Data are means � se (n � 23). ***P � 0.001. Scale bars � 20 �m.


structural nature of the ECM used in the study, since anECM constituted of native type 1 collagen has muchsmaller pore sizes, is less malleable (12), and has ahigher level of imprinting of plastic reorganization (59)than ECMs constituted of pepsin-extracted type 1 col-lagen in which the cross-links are broken during theextraction process. Indeed, a recent seminal work (14)has demonstrated an absolute requirement for theprotease-dependent process for tumor cells to invadenative type I collagen where its natural cross-linkedstructure more closely recapitulates the architecturalcharacteristics of in vivo ECM than does the pepsin-extracted type I collagen usually used and that lacks

these cross-links. Importantly, in this native collagen,low pH breaks the cross-links, mimicking the effect ofpepsin extraction but is reversibile (14). Given thisproteolytic requirement for tumor cell invasion to-gether with the use of invadopodia by aggressive cancercells as the structural framework for ECM digestion, itbecomes ever more important to understand invadopo-dial dynamics in the context of experimental systems/platforms of increasing similarity to in vivo tissues thatare involved in invasion. Indeed, a recent study (33)showed that MDA-MB-231 cells can enter Matrigel andform small membrane protrusions that are presentduring invasion, are sites of actin/cortactin coexpres-sion, and are enriched in invadopodial proteins. Sincefocal ECM digestion is still the only unambigous inva-dopodial marker, they were constrained to call thesestructures “invadopodia-like protrusions.” Here, the useof the in situ zymography fluorescence release protocolhas permitted the measurement of proteolysis in Matri-gel and both validate the results of Lizarraga et al. (33)and demonstrate that these structures are, indeed,invadopodia. Future studies need to be conductedon/in substrates that even more closely resemble the“natural” ECM composition. One interesting possibilityis the use of ex vivo explants (14) where cells fromprimary or secondary culture are injected into smallpieces of normal breast tissue from a resected tumorsample to directly monitor their ability to invade thetissue.

Up to now, in vivo detection of ECM proteolysis inclinical human cancer tissues has been limited to theanalysis of the level of degradation at low magnificationdue to the lack of reliable techniques to visualize

Figure 6. NHE1 is necessary for invadopodia proteolyticformation. To examine the role of NHE1 in invadopodiaformation, MDA-MB-231 cells were transfected with a smartpool of 100nM siRNA targeting NHE1 (NHE1 siRNA) or 100nM nonspecific siRNA as control (scrambled) siRNA (A) orthe transport dead NHE1 mutant, E266I (B). Two days aftertransfection, cells were plated on Matrigel with DQ-Green-BSA, and 24 h later, ECM digestion and cortactin or HA

(exogenous E266I NHE1) structures were analyzed in confocal microscopy, respectively. Cytofluorogram and ICA plots ofcolocalization using the JACoP image analysis plugin in ImageJ are shown next to the confocal expression images anddemonstrate a highly significant colocalization of cortactin and digestion in the cell treated with scrambled siRNA(ICQ�0.381), while the cell transfected with siRNA against NHE1 or with the E266I NHE1 mutant displayed hour-glasscurves showing no significant colocalization of digestion with either cortactin (ICQ�0.023) or HA (ICQ��0.018)expression, respectively. Scale bars � 10 �m.

Figure 7. Cellular focal ECM proteolytic ability is tightlyassociated with cellular expression of NHE1. MDA-MB-231cells were plated on Matrigel with DQ-Green-BSA; 24 h later,ECM digestion and NHE1 expression levels were analyzed inconfocal microscopy for a series of individual cells. NHE1expression levels and focal proteolytic levels for a series ofcells are shown plotted against each other (y�18.9�1.92x;r2�0.67; n�49, P�0.01).


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invadopodia in a tumor and to the inherent limits ofthe traditional technique of visualizing proteolysis viathe removal of background fluorescence (38). Re-cently, primary cells isolated from human head andneck squamous cell carcinoma or glioblastoma multi-forme were shown to focally degrade fibronectin orlaminin, respectively, at sites of invadopodia (59–61).However, the focal matrix digestion and invadopodialprotein expression analyses of these cells were per-formed in vitro outside of the tumor and, therefore, didnot resolve this question. In the present study, weapplied the new fluorescence release-based techniqueto frozen tissue sections from a breast cancer patientand observed, for the first time, precise areas of in vivoECM proteolysis that colocalize closely with NHE1expression directly in a tumor lobule from a patient’sbiopsy. If, in a larger study, the level of in vivo proteol-ysis will turn out to be highly correlated with clinicalinvasive behavior and poor prognosis, as we believe itwill be, then the clinical standardization of this analysiscould become both a prognostic marker and a predic-tive marker of anti-NHE1 therapy.

In further analogy with osteoclastic podosomes,where experimental extracellular acidificationstrongly increases osteoclast podosome proteolyticactivity (62, 63) by directly activating osteoclasticproteolytic programs such as NFATc1 accumulationvia elevation of intracellular calcium (54, 55), weobserved that exposure to a similar experimentalacidosis increased focal ECM degradation in MDA-MB-231 cells while having no significant effect onpericellular ECM digestion. This increased focal pro-teolysis occurred via a direct increase in invadopodialactivity and morphology as exposure to extracellularacidification produced a significant increase in bothtotal invadopodia number and length observed in thecell. Further, the rate of reacidification of the peri-invadopodial space of an invadopodia after an alka-linization was correlated with the initial, steady statepHe of that invadopodia and there was a relativelystable lag-period after alkalinization and the start ofthe reacidification. Both these phenomenona suggestthe presence of a pH receptor system similar to thatshown to regulate the osteolytic program in responseto low pHe in osteoclasts (54, 55) and to be presentin osteosarcoma (64) and in medullablastoma cells(65). It is intriguing to consider that mechanismsthat finely tune matrix proteolysis by either increas-ing or limiting acidification may be necessary foroptimal invasive motility (23). Which proton recep-tors are present in invadopodia and how their activitymight be modulated by other receptors such asmembers of the protease-activated receptor family inthese pH-sensitive invasive programs will be impor-tant future research directions.

The present study brings new insight into the mech-anisms regulating invasion in both a 2-D and 3-D matrixenvironment and provides a structural basis for thewell-known role of the NHE1 in tumor cell invasion andthe regulation of protease activity. It defines the roles of

the NHE1 in the regulation of invadopodia formationand in their subsequent digestion of the ECM and,consequently, of invasion in tumor cells. The preferen-tial localization of this invasion-specific protein modulein the cell membrane structure specific for invasion,the invadopodia, contributes further to the identifica-tion of a biochemical/structurally determined complexfundamental for the digestion and infiltration of sur-rounding tissues by the cancer cell. An importantfuture aspect to study will be to determine the mecha-nisms underlying the selection of the specific NHE1molecules to be trafficked or segregated to the inva-dopodia. This includes its phosphorylation and/orglycosylation status, its inclusion in specific types oflipid rafts, and its inclusion in specific protein-proteinsignal complexes. Furthermore, we believe that it willbe important to determine how changes in the stromaland in the metabolic microenvironments affect theactivity of the invadopodia and the status of NHE1 inrelation to the above characteristics. A major promiseof the study of the dynamics of invadopodial develop-ment and function has been the potential for dissectionof invasive mechanics with consequent identification ofnovel treatment mechanisms. In this light, the NHE1-dependent generation of a well-defined acidic pHe atinvadopodia that is essential for invadopodial proteol-ysis of the ECM identifies NHE1 as a strategic target fortherapeutic intervention.

The authors thank the Italian Association for CancerResearch (grant 5167) for supporting this work. The authors’laboratory is part of the Italian network Istituto NazionaleBiostrutture e Biosistemi. The authors thank Prof. DianeBarber (University of California, San Francisco, CA, USA) forkindly supplying the construct containing the ion transportdead E226I-NHE1 mutant and Sanofi-Aventis (Paris, France)for supplying cariporide (HOE642). The authors also thankDr. Roberto Buccione (Consorzio Mario Negri Sud, SantaMaria Imbaro, Italy) for teaching the basics of the invadopo-dia isolation technique.

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Received for publication December 3, 2009.Accepted for publication May 27, 2010.


NHE1 promotes invadopodial ECM proteolysis through acidification of the peri-invadopodial space

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