Functional proteomic screens reveal an essential extracellular role for hsp90α in cancer cell...

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NATURE CELL BIOLOGY VOLUME 6 | NUMBER 6 | JUNE 2004 507

Functional proteomic screens reveal an essentialextracellular role for hsp90α in cancer cell invasivenessBrenda K. Eustace1, Takashi Sakurai1,2, Jean K. Stewart1, Dean Yimlamai1, Christine Unger3, Carol Zehetmeier3,Blanca Lain3, Claudia Torella3, Stefan W. Henning3, Gerald Beste3, Bradley T. Scroggins4, Len Neckers4,Leodevico L. Ilag3 and Daniel G. Jay1,5

Tumour cell invasiveness is crucial for cancer metastasis and is not yet understood. Here we describe two functionalscreens for proteins required for the invasion of fibrosarcoma cells that identified the molecular chaperone heat shockprotein 90 (hsp90). The hsp90α isoform, but not hsp90β, is expressed extracellularly where it interacts with the matrixmetalloproteinase 2 (MMP2). Inhibition of extracellular hsp90α decreases both MMP2 activity and invasiveness. This rolefor extracellular hsp90α in MMP2 activation indicates that cell-impermeant anti-hsp90 drugs might decrease invasivenesswithout the concerns inherent in inhibiting intracellular hsp90.

Cancer invasiveness is a complex process with three prominent stages:adhesion to the extracellular matrix, digestion of the matrix to releasecells from the tumour mass, and migration of the tumour cell to sec-ondary targets1. Current post-genomic strategies are being applied toidentify genes and their encoded proteins that function in tumourinvasion. For example, microarray analyses have identified differen-tially expressed genes from many classes that might contribute to inva-siveness2–6, but these expression changes only suggest functionalimportance. RNA-mediated interference (RNAi) screens in mam-malian cells can functionally assess the genome in high throughput,and these screens for cancer phenotypes have identified new gene tar-gets7,8. However, such screens are limited for the following reasons:RNAi knockdown occurs over many hours, thus the screens might besusceptible to compensation mechanisms, and proteins that turn overslowly cannot be efficiently depleted. A high-throughput approachthat directly addresses protein function and is temporally restrictedcould identify new and different targets for tumour invasion or otherdisease-relevant processes.

Towards this end, we developed and performed two proteomicscreens to identify and validate proteins required for cancer invasion.These screens used fluorophore-assisted light inactivation (FALI)9

for acute protein knockdown directed by specific binders to compo-nents of the surface proteome with monoclonal antibody libraries orrecombinant phage display antibodies10. FALI is a high-throughputmethod of damaging proteins in situ by using fluorescein-labelledbinders and 490-nm diffuse light. FALI-treated cells were then

assayed for invasiveness. Antibodies that recognize proteins criticalfor invasion were then used for immunoprecipitation and mass spec-trometry to identify the target protein. We focused our screens on thesurface proteome as an accessible subset of the proteome that wouldprovide good targets for drug discovery.

Both screens identified the molecular chaperone hsp90 as animportant extracellular mediator of invasion. We further showedthat the hsp90α isoform, but not hsp90β, is expressed extracellularlyon fibrosarcoma and breast cancer cells. Hsp90α interacts withMMP2 outside the cell and promotes MMP2 activation, which iscritical for tumour invasiveness.

RESULTSScreening for tumour invasion proteinsWe performed two independent screens for surface proteins that func-tion in tumour cell invasion. In the first, we used a library of 1,152monoclonal antibodies (mAbs) raised against HT-1080 fibrosarcomacells, and selected 19 that bound to the cell surface (Fig. 1). The secondscreen used a recombinant phage display single-chain variable frag-ment (scFv) binder library (107 phages) prepared from spleen mRNAderived from mice immunized with HT-1080 cells. This library is com-posed of phages that express scFv recombinant proteins representingthe antigen-binding repertoire of the immunized mice. We screenedthis library for scFvs that bound preferentially to HT-1080 surfaceantigens in comparison with a control Hs27 fibroblast cell line, andselected 13 scFvs for further functional analysis (Fig. 1). We performed

1Department of Physiology, Tufts University School of Medicine, Boston, MA 02111, USA. 2Present address: RIKEN Brain Science Institute, 2-1 Hirosawa, Wako,Saitama 351-0198, Japan. 3Xerion-Pharmaceuticals AG, Sauerbruchstrasse 50, D-81377, Munich, Germany. 4Cell and Cancer Biology Branch, National CancerInstitute, Rockville, MD 20850, USA.5Correspondence should be addressed to D.G.J. (e-mail: [email protected]).

Published online: 16 May 2004; DOI:10.1038/ncb1131

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© 2004 Nature Publishing Group

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508 NATURE CELL BIOLOGY VOLUME 6 | NUMBER 6 | JUNE 2004

FALI with these two sets of binders and assayed the resulting cellsamples for invasiveness across a Matrigel-coated membrane, asdescribed previously9. One of the 19 monoclonal antibodies(Fig. 2a), and six of the 13 scFvs (Fig. 2b) showed a significant inhi-bition (P < 0.05, t-test) of invasion.

Immunoprecipitation and mass spectrometry were used to identifythe cognate antigens of the binders after functional screening11,12.Some proteins identified in the scFv screen (Table 1), such as α2β1-integrin and α3β1-integrin, have established roles in adhesion,

migration and invasion (reviewed in ref. 13). The fact that these pro-teins were identified multiple times by our screen strengthens their roleas important cancer therapeutic targets and serves as a proof of princi-ple for this screen. It is interesting to note also that one of the six scFvsthat inhibited invasion was function-blocking even without FALI treat-ment (IID3; C.U., unpublished data). This is a significant observationbecause neutralizing scFvs provide a direct route to humanized anti-bodies that might serve as therapeutic agents for the treatment ofmetastatic cancer. FALI was able to convert five of the

HT-1080 fibrosarcoma cells Inject mice

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Figure 1 Generation of HT-1080 cell surface binders. Mice wereinoculated with fixed or lysed HT-1080 cells. Hybridomas were generatedfor mAbs, and phage display libraries were generated fromimmunoglobulin genes (VH and VL) to generate recombinant scFvs. In all,1,152 mAbs and 107 phages were isolated. The 1,152 mAbs werescreened by permeabilized immunofluorescence and 218 were found tobind to HT-1080 cells. Of those, 19 bound to the surface of HT-1080cells. The 107 phages were panned against HT-1080 cells, and 2,760

phages were isolated. With the use of binding ELISA, 155 of theresulting scFvs were selected as HT-1080-specific binders. The 155scFvs were screened again by binding ELISA to select scFvs withincreased surface expression on HT-1080 cells compared with Hs27fibroblasts (more than twofold increase). A total of 44 scFvs were isolatedand their VH and VL cDNAs were sequenced; 13 unique scFvs wereselected for FALI-invasion assays, and their increased binding to HT-1080 cells compared with Hs27 cells was confirmed by FACS.

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non-function-blocking scFvs into function-blocking reagents, demon-strating the enhancement that FALI provides in these screens.

Both screens identified hsp90 (Table 1), a molecular chaperone thatbinds and stabilizes substrate proteins; by doing so it promotes proper

activity and function14. Typical substrates of hsp90 include a variety ofkinases and phosphatases, and also nuclear hormone receptors15. Infact, all previously reported substrates of hsp90 are intracellular pro-teins, and it has been primarily described as an intracellular molecular

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Figure 2 FALI screening of HT-1080 cell surface binders. (a) Nineteen surface-binding monoclonal antibodies (listed along thebottom) were screened in a FALI invasion assay (10 µg ml−1). Oneantibody, 1.5.1, shows a significant inhibition of invasion after exposure tolight (asterisk, P < 0.05, t-test). The negative control (0, no antibody) doesnot significantly affect invasion, whereas the positive control anti-β1integrin shows a significant inhibition of invasion after exposure to light

(JB1; asterisk, P < 0.05, t-test; 10 µg ml−1). (b) Thirteen surface-bindingscFvs (listed along the bottom) were screened in the FALI invasion assay(20 µg ml−1). Six scFvs show a significant (asterisk, P < 0.05, t-test)inhibition of invasion after exposure to laser light. Anti-β1-integrin (JB1;20 µg ml−1), and the negative control (0, no antibody) are also shown. Inboth panels, each bar is normalized to the negative control and representsthe mean ± standard error from at least two experiments in triplicate.

Table 1 FALI-induced inhibitors of tumour invasion from the screen identified by immunoprecipitation and mass spectrometry

Screen identifier Protein identity MW Mr (K) Matched peptides (#) Sequence coverage (%)

mAb 1.5.1 Hsp90α 90 16 24

Hsp90β 90 24 33

α-actinin-4 105 13 20

scFv IIIF1* Hsp90α 90 26 26

Hsp90β 90 18 27

scFvs G6, IC7, Integrin α2 >150 12–26 12–22

IB4, IH5, IID3 Integrin β1 130 7–15 9–16

scFvs ID6, IIC1, Integrin α3 130 10–13 9–13

IIB2, IIIG7, IIIC6, IIIE4 Integrin β1 130 7–11 9–13

*There was some cross-reactivity with integrin α2.

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chaperone. Our identification of hsp90 was therefore surprisingbecause our screens are targeted specifically to the surface proteome.

Extracellular localization of the hsp90α isoformTo confirm the MS identification and extracellular location of hsp90,we performed immunoprecipitation, surface biotinylation andimmunocytochemistry. Immunoblotting with an hsp90-specific anti-

body showed that hsp90 was the immunoprecipitated protein from ourpositive result in the monoclonal antibody screen (1.5.1) (Fig. 3a). Weshowed that hsp90, but not α-actinin-4, is on the surface of HT-1080cells by surface biotinylation16 (Fig. 3b). We used α-actinin-4 as a con-trol because 1.5.1 also immunoprecipitated it (Table 1) and its intracel-lular function might be important for invasion17.Immunocytochemistry using non-permeabilized HT-1080 cells

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Figure 3 Hsp90α is an extracellular protein on HT-1080 cells and inconditioned medium. (A) Immunoprecipitation (IP) of HT-1080 cell lysates wasdone with beads alone, with 1.5.1 or with anti-hsp90 antibodies. The sameprotein was recognized by immunoblot (blot) analysis with antibodies specificfor either 1.5.1 or hsp90. (B) Surface proteins were biotinylated andimmunoblotted with either anti-hsp90 or anti-α-actinin-4. Unbiotinylatedproteins represent the intracellular pool not exposed to the biotinylationreagent. Separation of the biotinylated surface proteins (Surface) from theunbiotinylated intracellular pool (Intracellular) was done with magneticstreptavidin beads. 1.5.1 and scFv IIIF1 also show surface and intracellularlocalization (data not shown). (C) Surface proteins were detected byimmunocytochemistry on non-permeabilized HT-1080 cells with the following

antibodies: mouse secondary alone (a), a rabbit secondary alone (b), anti-β1-integrin (c), anti-α-actinin-4 (d), anti-hsp90α (e) and anti-hsp90β (f). MDA-MB231 breast cancer cells are shown for anti-hsp90α (g) and anti-hsp90β (h).Secondary antibodies were labelled with alkaline phosphatase. Anti-β1-integrinand anti-hsp90α antibodies showed robust surface staining, whereas thesecondary antibody controls, anti-α-actinin-4, and anti-hsp90β showed nodetectable surface localization. MDA-MB231 controls showed similar staining(see Supplementary Information, Fig. S1). Images were made with an OlympusBH-3 and were collected with a SPOT digital camera (Diagnostic Instruments,Sterling Heights, MI). Scale bar, 10 µm. (D) Serum-free conditioned medium(concentrated) and total cell lysate (50 µg) from HT-1080 cells wereimmunoblotted with anti-hsp90α and anti-hsp90β antibodies.

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showed that the inducible hsp90α isoform, but not hsp90β, is found onthe surface of HT-1080 cells (Fig. 3c). We also observed surface expres-sion of hsp90α on a different invasive cell line, MDA-MB231 breastadenocarcinoma cells, showing that hsp90α surface expression is notrestricted to HT-1080 cells (Fig. 3c; Supplementary Information,Fig. S1). Immunoblotting of HT-1080 conditioned medium withhsp90α-specific and hsp90β-specific antibodies also showed thathsp90α, but not hsp90β, is found in the extracellular space (Fig. 3d).

It is unclear how hsp90α is secreted, but there is precedent for itssecretion from vascular smooth muscle cells after oxidative stress18. It isnot likely that hsp90α arises from lysed cells in culture because wewould then expect to observe as much of the β isoform as we do of theα. Although hsp90α does lack a classical signal sequence, there aremany extracellular proteins that lack obvious signal sequences, such asfibroblast growth factor-2 (ref. 19), which is also secreted through an

unknown mechanism. One possibility is that hsp90α might be releasedthrough exosomes from tumour cells, as observed with B cells20 anddendritic cells21.

Hsp90α mediates fibrosarcoma invasionTo establish hsp90’s role in invasion, we performed FALI with antibod-ies specific for hsp90 and used pharmacological inhibitors of hsp90. Weobserved a dose-dependent decrease in invasion of HT-1080 cells afterFALI with 1.5.1, antibodies against hsp90α-β and hsp90α, but notagainst hsp90β or α-actinin-4 (Fig. 4a). FALI of 1.5.1 or hsp90α alsocaused a significant inhibition of invasion in MDA-MB231 cells(Fig. 4b), indicating that the role of extracellular hsp90α in tumourinvasion might apply to multiple tumour models. Pharmacologicalinhibitors of hsp90 also inhibited invasion. The amino-terminal hsp90inhibitor geldanamycin, and the carboxy-terminal hsp90 inhibitorscoumermycin A1 and novobiocin, significantly inhibited invasion(P < 0.01, ANOVA; Fig. 4c). In contrast, nalidixic acid, which inhibits

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Figure 4 FALI with anti-hsp90 antibodies and pharmacological agentsinhibit invasion. (a) Antibodies specific for hsp90, hsp90α, hsp90β, α-actinin-4 and 1.5.1 were labelled with FITC and assayed for invasivenessafter FALI at three concentrations (10, 20 and 40 µg ml−1) with HT-1080cells. Antibodies against 1.5.1, hsp90 and hsp90α showed concentration-dependent inhibition of invasion after FALI, whereas antibodies againsthsp90β and α-actinin-4 did not. The positive control, anti-β1-integrin (20µg ml−1), and the negative control (0, no antibody) are also shown. Each baris normalized to the dark control and represents the mean ± standard errorfrom at least two experiments in triplicate. (b) An antibody specific forhsp90α and 1.5.1 were FITC-labelled and assayed for invasiveness afterFALI (20 µg ml−1 antibody). Antibodies against 1.5.1 and hsp90α showedinhibition of invasion after FALI in MDA-MB231 breast adenocarcinomacells. Each bar is normalized to the dark control and represents the mean ±standard error from at least two experiments in triplicate. (c) HT-1080 cellswere pretreated for 1–2 h with vehicle (dimethyl sulphoxide or water, whichgave similar results), hsp90 inhibitors [10 µM geldanamycin (GA), 100 µMcoumermycin A1 (CM), 500 µM novobiocin (NV)) or a control inhibitor (500µM nalidixic acid (NA)) and assayed for invasiveness. Treatment with GA,NV and CM showed a significant inhibition of invasion (P < 0.05, ANOVA).Viability of cells in this assay was not affected (data not shown). Each datapoint is normalized to the no drug control and represents themean ± standard error from a representative experiment in triplicate.

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Figure 5 Hsp90α interacts with and mediates MMP2 activity extracellularly.(a) Hsp90α or hsp90β was immunoprecipitated (IP) from HT-1080conditioned medium and immunoblotted (blot) with anti-MMP2 or anti-hsp90α. (b) HT-1080 cells were treated with geldanamycin (GA, 20 µM) inserum-free media and assayed by zymography. The only digested area seencorresponds to MMP2 (72 kDa gelatinase) activity and shows a decrease inactivity after treatment with GA. Enzymatic activity was quantified bydensitometry, and the graph shows a significant decrease in MMP2 activity(asterisk, P < 0.01, t-test). Total protein content in conditioned medium wasrevealed by silver staining to ensure equal loading (data not shown). (c) HT-1080 cells were left untreated or treated with 10 µM geldanamycin (GA) for1 h. Activated MMP2 (aMMP2; 100 ng) enzyme was then added to half ofthe samples for 30 min and the samples were then assayed for invasiveness.GA caused a significant decrease (asterisk, P < 0.05, t-test) of invasion thatwas eliminated when activated MMP2 was added. Data points arenormalized to the corresponding no-drug control and represent themean ± standard error from a representative experiment in triplicate.

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the DNA topoisomerase II complex22 (a secondary target of novo-biocin and coumermycin A1), but not hsp90, had no effect on invasion.Together these independent validation models of FALI and pharmaco-logical inhibition demonstrate the role of hsp90α in invasion.

MMP2 is a target of extracellular hsp90αHow might extracellular hsp90α act in invasion? Although hsp90 hasmany well-established intracellular functions (reviewed in refs 15, 23),reports of extracellular function are scarce24–27. One hypothesis wetested was that hsp90 could act as a component of the receptor for thechemoattractant lipopolysaccharide (LPS)28 to promote invasion. Ourresults do not support this hypothesis because HT-1080 cells were onlyweakly responsive to LPS in the invasion assay, and there was no signifi-cant difference between FALI-treated, drug-treated and untreated cellsin their response to LPS (see Supplementary Information, Fig. S2).

Instead, our findings suggest that hsp90α acts as an extracellularmolecular chaperone. One well-characterized property of invasivetumours is their ability to accelerate the degradation of extracellularmatrix by MMPs29. The degradation of the surrounding extracellularmatrix and tissue by MMPs provides access to the vasculature and lym-phatic system, allowing tumour dissemination. MMPs have increasedexpression and activation in almost all human cancers30, and invasiveHT-1080 cells are known to express active MMP2 (ref. 31). Pro-MMPsare secreted and activated extracellularly by proteolytic digestion of theinhibitory propeptide domain32. On the basis of the known role andimportance of hsp90 in activation of intracellular client proteins33, weproposed that extracellular hsp90 might have a role in the activation ofMMPs outside the cell.

Several lines of evidence support a role for extracellular hsp90α inMMP2 activation. First, we showed that the matrix metalloproteinaseMMP2 co-immunoprecipitates with hsp90α from HT-1080-condi-tioned medium (Fig. 5a), showing a direct interaction between theseproteins extracellularly. Second, we showed that inhibition of hsp90affected MMP2 activity. We applied an hsp90 inhibitor, geldanamycin,to HT-1080 cells to test whether inhibition of hsp90 decreased MMP2activity. Secreted MMP2 activity, assayed by zymography31, wasdecreased significantly by geldanamycin treatment in comparison withuntreated controls (P < 0.01, t-test; Fig. 5b). Third, we showed that thedecrease in MMP2 activity caused by hsp90 inhibition is directlyresponsible for the loss of invasiveness, because we can rescue the lossof invasion by adding back activated MMP2 (Fig. 5c).

However, geldanamycin can inhibit hsp90 inside the cell. To showthat extracellular hsp90 is critical for invasiveness and MMP2 activa-tion, we used extracellularly restricted geldanamycin beads (GA-beads)34. GA-beads had no effect on intracellular functions of hsp90,such as Akt phosphorylation35 or Akt and Raf expression36, comparedwith control beads (Fig. 6a). In contrast, GA-beads, but not controlbeads, inhibited HT-1080 invasion by 45%, showing the importance ofextracellular hsp90 in invasiveness (Fig. 6b; P < 0.01, t-test). The GA-beads also caused an 80% decrease in the active MMP2 protein, butonly a 15% decrease in pro-MMP2 protein, in extracellular medium(Fig. 6c, Supplementary Information, Fig. S3). This supports a role forextracellular hsp90 in the activation of MMP2 as opposed to secretion,because pro-MMP2 protein levels show only a modest change. In addi-tion, most MMP enzymes, including MMP2, are released through theconstitutive secretory pathway after translation, and direct regulationof this pathway is not common32. We cannot rule out a role for hsp90 instabilization of active MMP2, but this seems less likely given its well-characterized role in maturation of intracellular proteins35,36.

DISCUSSIONThis ‘function first’ strategy to assess proteins required for invasivenessis scalable and might provide the opportunity for a global interrogationof protein function by using larger binder libraries. This strategy hasalready revealed an extracellular role for hsp90α that might be clinicallyimportant. It is known that hsp90 has a significant role in cancer37, and

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Figure 6 Extracellular inhibition of hsp90α decreases invasion and activeMMP2 protein. (a) HT-1080 cells were treated with no beads, 10% controlbeads or 10% GA-beads (v/v) for 6 h at 37 °C under 7% CO2. Cells werelysed; 50 µg of protein was loaded in each lane and immunoblotted with theindicated antibodies. No change in phospho-Akt, total Akt, Raf-1 or hsp90αprotein levels is seen, which is consistent with the extracellular restriction ofthe GA-beads. (b) HT-1080 cells were treated either with no beads or with5% or 10% (v/v) GA-beads or control beads and assayed for invasiveness.GA-bead-treated cells show a significant decrease in invasion(asterisk, P < 0.01, t-test) compared with control-bead-treated cells. Eachdata point is normalized to the no-treatment control and represents themean ± standard error from two triplicate assays. (c) HT-1080 cells weretreated with GA-beads or control beads (beads) or left untreated (0).Conditioned medium was immunoblotted (blot) for MMP2 or hsp90α. ActiveMMP2 levels were decreased by 80%, whereas pro-MMP levels were lessaffected (15%) after treatment with GA-beads. For a full view of the gelsused to construct this figure, see Supplementary Information Fig. S3.

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there is growing evidence that hsp90α expression in particular iscrucial38–40. Here we show that extracellular hsp90α is required forinvasiveness in two distinct cancer cell lines, indicating that this rolemight be common among tumours. Recent studies show that hsp90from tumour cells binds ATP with high affinity (39 ± 14 nM) in com-parison with normal cells41 and is therefore likely to be active in theextracellular environment of tumours, where ATP concentrations are inthe low micromolar range42. We therefore propose that extracellularhsp90α might act in vivo to increase cancer invasiveness by promotingthe activation of MMP2 to increase the digestion of extracellular matrixproteins and thus to enhance metastasis.

Hsp90 action on the cell surface might be a wide-ranging phe-nomenon. Hsp90 has also been implicated in peptide antigen pres-entation to macrophages and dendritic cells43. This, coupled withour findings, suggests the possibility that hsp90α chaperones abroad array of proteins and peptides in the extracellular space thatact in a variety of cellular processes.

Early clinical trials of MMP inhibitors have been disappointingbecause of a lack of tumour specificity, and current strategies are nowfocused on the regulatory mechanisms of MMP expression and activa-tion44 in tumour cells. Although clinical trials are underway for hsp90inhibitors, such as the geldanamycin derivative 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG)37, a more compartmentalizedapproach to hsp90 inhibition might be beneficial to patients. Our find-ings suggest a new therapeutic strategy in which extracellular hsp90α istargeted to decrease the activity of MMPs in invasive tumours withoutaffecting the broad intracellular function of this molecular chaperone.

METHODSCells and reagents. All cells were obtained from ATCC (Manassas, VA), namelyHT-1080 fibrosarcoma cells (CCL-121), Hs-27 fibroblasts (CRL-1634) and MDA-MB231 adenocarcinoma cells (HTB-26). Antibodies used were as follows: anti-hsp90α-β (SPA-830; Stressgen, Victoria, BC, Canada), anti-hsp90α (PA-013;Affinity Bioreagents, Golden, CO), anti-hsp90β (PA-012, Affinity Bioreagents),anti-β1-integrin (MAB2253Z; Chemicon, Temecula, CA), anti-α-actinin-4 anti-body (gift from Martin Pollak), anti-Akt (9272; Cell Signaling, Beverly, MA) andanti-phospho-Akt (9277; Cell Signaling), anti-Raf-1 (KAP-MA020; Stressgen);antibodies conjugated to fluorescent markers and to alkaline phosphatase(Molecular Probes, Eugene, OR). Geldanamycin and activated MMP2 enzymewas obtained from Chemicon. GA-beads were prepared as described previously34.

Labelling with fluorescein isothiocyanate, and FALI invasion assay. Fluorescein isothio-cyanate (FITC) labelling of mAbs and scFvs was done as described previously9. The inva-sion assay was performed with fluorescently labelled cells (for mAbs, CellTracker Orange(Molecular Probes);for scFvs,bisbenzimide H (Sigma,St Louis,MO)) that were incubatedwith FITC-labelled mAbs (purified using T-gel (Pierce (Rockford, IL)) (10 µg ml−1) orscFvs (20 µg ml−1).Halfof the mAb-treated cells were exposed to 1 h of300-W filtered lighton ice (Brilliant Blue no.69 (Roscolux,Stamford,CT)) and the other half were kept in thedark on ice.Of the scFv-treated cells,half were exposed to 90 s of 0.6-W continuous-wave488-nm laser light on ice,and the other half were kept in the dark on ice.After a 6-h incuba-tion at 37 °C under 7% CO2,the number of cells that invaded a basement membrane bar-rier (0.3 µg µl−1 Matrigel (BD Biosciences, Bedford, MA)) on a porous membrane (poresize 8 µm (Neuroprobe, Gaithersburg, MD)) were quantified in each well with a fluores-cence plate reader (for CellTracker Orange, excitation was at 544 nm and emission was at590 nm;for bisbenzimide H,excitation was at 370 nm and emission was at 460 nm).

Monoclonal antibody library construction. Mice were immunized with fixed andlysed HT-1080 cells, and mouse hybridoma cultures were generated as describedpreviously11. For surface protein immunocytochemistry, HT-1080 cells were incu-bated with hybridoma supernatant, then fixed with paraformaldehyde. Secondaryand tertiary fluorescent antibodies were used to visualize antibody staining.

ScFv library construction. Total RNA was isolated from spleens of mice immu-nized with HT-1080 cells. Complementary DNA was synthesized by using aprimer mix to amplify the immunoglobulin constant and variable regions.

Polymerase chain reaction products were cloned into the phage display vectorpXP10. The ligation mix was transfected into Escherichia coli TG-1 by electropo-ration, resulting in a library size of 107 independent clones.

Selection and screening of scFv antibodies. HT-1080 cells were harvested andfixed with paraformaldehyde onto wells of a 96-well ultraviolet cross-link plate(Corning Inc., Acton, MA) blocked with sodium metaperiodate. 1012 colony-forming units (cfu) of phage library per 106 cells was added. Bound phages wereeluted with 10 mM glycine pH 2.2, and neutralized with 1 M Tris-HCl pH 7.4 togenerate 103–106 cfu. This eluate was amplified by infecting liquid culture (E.coli TG1). Phagemid-containing E. coli were then propagated by overnightgrowth on Luria–Bertani agar plates containing 100 µg ml−1 ampicillin and 1%glucose. Phage particles for the next round of selection were produced by super-infecting cultures from the previous round of selection with helper phage VCS-M13 (Stratagene, La Jolla, CA) and growing the cultures overnight at 20 °C in2×TY containing 100 µg ml−1 ampicillin and 50 µg ml−1 kanamycin. Selection-ready phages were precipitated from the cleared bacterial supernatant with 0.5M NaCl containing 4% PEG-6000.

The genes encoding the selected scFv contained in the phage display vectorwere re-cloned in expression vector pXP14. The bacteria were grown at 30 °C in2×TY containing 100 µg ml−1 ampicillin and 0.1% glucose, and inducedovernight at 25 °C in 0.5 mM isopropyl β-D-thiogalactoside in 96-wellmicrotiter plates. scFv-containing cleared lysates were prepared by adding hen-egg lysozyme (50 µg ml−1) to the bacterial cultures, followed by centrifugationat 3000g. For the screening enzyme-linked immunosorbent assay (ELISA), HT-1080 cells were harvested, fixed with paraformaldehyde, diluted to 107 cells ml−1

in PBS and immobilized onto wells of a 96-well ultraviolet cross-link plate thatwere blocked with the scFv-containing blocked cleared lysates. The plates wereincubated with horseradish-peroxidase-conjugated anti-E-tag (AmershamBiosciences, Piscataway, NJ) and developed with peroxidase (Roche Diagnostics,Indianapolis, IN). Positive clones were retested against HT-1080 cells and con-trol Hs-27 human fibroblasts with the ELISA screening procedure as described.

Fluorescence-activated cell sorter (FACS) analysis with HT-1080 cells and Hs-27 cells (106 cells ml−1) was performed to validate differential surface binding.Cells were incubated with pure scFv (10 µg ml−1 in CellWash; BD Biosciences,Bedford, MA) and bound scFvs were detected with a secondary FITC-labelledanti-E-tag antibody. Samples were analysed on a Becton Dickinson FACSscan.

Immunoprecipitation and mass spectrometry. Immunoprecipitation of 1.5.1 wasdone essentially as described previously45, using ascites fluid derived from the 1.5.1hybridoma (10 µl per 1 mg cell extract) and anti-mouse IgG-agarose (Sigma).Immunoprecipitated proteins were analysed by SDS–polyacrylamide-gel elec-trophoresis (SDS–PAGE) and Coomassie staining. Stained bands were excised andsubjected to in-gel tryptic digestion. The trypsinized peptide fragments were runon a Surveyor HPLC and LCQ Deca Ion Trap mass spectrometer(ThermoFinnigan, Woburn, MA) with a 75 µm nanospray C18 column (NewObjectives, Woburn, MA). The peptide mass fragments obtained were used tosearch all entries for the species Homo sapiens in the NCBI and SwissProt databases.

For scFv immunoprecipitations, HT-1080-specific scFvs were coupled toStrepTactin Sepharose (50 µg per 50 µl resin) and the washed scFv beads wereadded to the cleared lysates (1 mg total protein). The scFv target complexes wereeluted with 10 mM D-desthiobiotin in PBS containing 0.1% Tween 20;immunoprecipitated proteins were analysed by SDS–PAGE and silver staining.Stained bands were excised and subjected to in-gel digestion with trypsin. Thepeptide fragments were extracted from the gel pieces and desalted on ZipTipµC18; the eluted peptides were spotted on a Teflon-coated matrix-assisted laserdesorption ionization (MALDI) target. The samples were analysed on a STR-DEVoyager MALDI mass spectrometer (Applied Biosystems, Foster City, CA) andthe peptide masses obtained were used for protein identification by peptidemass fingerprinting, searching all entries for the species Homo sapiens in theNCBI and SwissProt databases.

Immunocytochemistry. HT-1080 cells were incubated with primary antibodyin complete medium at 4 °C and then fixed. Alkaline-phosphatase-conjugatedsecondary antibodies were used and activity was detected with the Nitro BlueTetrazolium–5-bromo-4-chloroindol-3-yl phosphate substrate (NBT-BCIP;Roche Diagnostics, Piscataway, NJ) as directed.

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A RT I C L E S


Note: Supplementary Information is available on the Nature Cell Biology website.

ACKNOWLEDGEMENTSWe thank M. Berne for performing the mass spectrometry analysis on 1.5.1immunoprecipitates, and J. Dice, J. Kerner and K. Sloan for critical reading of themanuscript. This work was supported in part by grants from the National CancerInstitute (to D.G.J.), a National Institutes of Health Training grant (B.K.E.) and aNational Institutes of Health Program grant to the Gastroenterology Research onAbsorptive and Secretory Processes group (GRASP).

COMPETING FINANCIAL INTERESTSThe authors declare that they have competing financial interests: see Nature CellBiology website for details.

Received 15 January 2004; Accepted 19 April 2004Published online at http://www.nature.com/naturecellbiology

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