tumorcellinvasioncanbeblockedbymodulators of collagen ...published onlinefirst may 24, 2016; doi:...

Therapeutics, Targets, and Chemical Biology

TumorCell InvasionCanBeBlockedbyModulatorsof Collagen Fibril Alignment That ControlAssembly of the Extracellular MatrixMoran Grossman, Nir Ben-Chetrit, Alina Zhuravlev, Ran Afik, Elad Bassat,Inna Solomonov, Yosef Yarden, and Irit Sagi

Abstract

Abnormal architectures of collagen fibers in the extracellularmatrix (ECM) are hallmarks of many invasive diseases, includingcancer. Targeting specific stages of collagen assembly in vivopresents a great challenge due to the involvement of variouscrosslinking enzymes in the multistep, hierarchical process ofECM build-up. Using advanced microscopic tools, we monitoredstages of fibrillary collagen assembly in a native fibroblast-derived3Dmatrix system and identified anti-lysyl oxidase-like 2 (LOXL2)antibodies that alter the natural alignment and width of endo-

genic fibrillary collagens without affecting ECM composition. Thedisrupted collagen morphologies interfered with the adhesionand invasion properties of human breast cancer cells. Treatmentof mice bearing breast cancer xenografts with the inhibitoryantibodies resulted in disruption of the tumorigenic collagensuperstructure and in reduction of primary tumor growth. Ourapproach could serve as a general methodology to identify noveltherapeutics targetingfibrillary protein organization to treat ECM-associated pathologies. Cancer Res; 76(14); 4249–58. �2016 AACR.

IntroductionThe extracellular matrix (ECM) is a dynamic and versatile

biomaterial that regulates tissue integrity and cell–cell connec-tions, interactions, and communication (1). The ECM not onlyprovides structural support to the tissue but can also affect cell fateby altering its degree of stiffness, organization, and molecularcomposition (2–4). Therefore, ECM homeostasis is critical forcellular function, signaling, and development, and dysregulatedECM remodeling is a common driving factor in various patho-logic conditions (5, 6).

The ECM scaffold of each tissue is composed of specific fibrousand nonfibrousmacromolecules, proteoglycans, and glycoproteinsthat dictate the tissue's unique biophysical and mechanical prop-erties. Collagens are the major protein components of the ECM,and their synthesis and assembly are multistep, hierarchical pro-cesses that dictate the final structure andmechanical integrity of theECM (7). Fibrillary collagens are assembled into precisely alignedsuprastructures (microfibrils, fibrils, fibers) by intramolecular andintermolecular covalent crosslinking, catalyzed by lysyl oxidase(LOX) enzymes. The copper-dependent LOX family consists ofLOX and four LOX homologs (LOXL1, LOXL2, LOXL3, andLOXL4; ref. 8). The crosslinks produced by LOX enzymes endow

collagen fibrils and other supramolecular assemblies with tensilestrength and mechanical stability required for tissue functionality.

LOX expression is often associated with specific tissues andbiologic processes (9), and dysregulated expressionwas identifiedin many pathologic states (10–12). Elevated LOX expression islinked to the disrupted collagenmorphology and increased tissuestiffness often found in tumors, leading to aberrant cellularbehavior (10, 13). Activation of LOXs during cancer was alsodemonstrated to affect intracellular signaling pathways by mod-ification of snail, (14) integrins (6), and repression of E-cadherin(15), leading to induction of epithelial-to-mesenchymal (EMT)transition. Among the LOX family, LOX and LOXL2 are highlyexpressed in many human cancers, partly and adversely in corre-lation with clinical outcome (10). Overexpression of LOXL2 innormal epithelial cells induced aberrant acinar morphogenesis(16). In the tumor stroma, LOXL2 mediates fibroblast activationthrough integrin engagement and FAK signaling (17). Geneticdepletion of both LOX and LOXL2 in various animal models forcancer resulted in impeded disease progression and reduction inmetastasis (18, 19). Inhibition of LOX or LOXL2 by mAbs pre-vented fibrosis-enhanced metastatic colonization (20) and atten-uated the disease in various models of cancer (18, 21). Theseresults proposed that LOXand LOXL2 are key players in the tumormicroenvironment and serve as therapeutic targets in the clinic.

Although both LOX and LOXL2 clearly play major roles intumor progression, it has been proposed that these enzymescannot compensate for each other and have distinct roles in vivo(18). Loss-of-function animal models for LOX and LOXL2 dem-onstrated differential tissue dysfunctions indicating the enzymes'different roles in development and homeostasis (22–24). Inaddition, overexpression of these enzymes in cancer has beencorrelated with distinct events during tumor progression. LOXL2is highly overexpressed in invasive metastatic tumors comparedwith noninvasive ones (12) and is linked to promoting tumor cellinvasion and angiogenesis (18, 25). However, the exactmolecular

Department of Biological Regulation,Weizmann Institute of Science,Rehovot, Israel.

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

M. Grossman and N. Ben-Chetrit contributed equally to this article.

Corresponding Author: Irit Sagi, Weizmann Institute of Science, 234 Herzl St.,Rehovot 76100, Israel. Phone: 972-8934-2130; Fax: 972-8934-4145; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-15-2813

�2016 American Association for Cancer Research.

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mechanisms by which these enzyme execute their enzymaticactivities and how they contribute to tumor malignancy is notfully understood,mainly due to the complexity of ECMmoleculesand the technical challenges associated with structurally charac-terizing these molecules in high resolution. Currently, informa-tion regarding the effect of LOX inhibition on ECM morphologyhas been demonstrated in several studies (6, 20, 26), while themolecular insights into the role of LOXL2 in collagen assembly innormal and pathologic scenarios has yet to be determined.

Therefore, we set to screen formodulators of collagen assemblytargeting LOXL2, to both interfere with pathologic collagenassembly, as well as to gain new molecular insights into thefunction of LOXL2 in the ECM. We established a system tomonitor changes in ECM assembly brought about by activatedstromal cells and identified a high-affinity clone of anti-LOXL2mAb capable of specifically altering fibrillary collagen alignment.Disrupting ECM alignment negatively affected cancer cell migra-tion, adhesion, and invasion in vitro. Our findings point to thepotential therapeutic benefit to be gained by disrupting LOXL2-mediated ECM fibril alignment, as demonstrated in a mousemodel of breast cancer, as a complementary therapeutic approachto conventional treatment. Our results reveal the specific contri-bution of LOXL2 to fiber alignment during the final stages ofcollagen crosslinking, thus serving as a key mediator of tumorprogression.

Materials and MethodsLOXL2 expression and purification

The catalytic domain of human LOXL2 (545–775) was clonedinto pET28 expression vector and transformed into competentEscherichia coli BL21. The inclusion bodies fraction was resus-pended in buffer A (see Supplementary Table S1) at 4�C, soni-cated, and centrifuged at 9,000 rpm, and the procedure wasrepeated in buffer B. Solubilization of the pellet in buffer C wasfollowed by anion exchange using bufferD and a gradient ofNaClconcentration. The collected protein-containing fractions werediluted <50 mg/1 mL in buffer E and refolded by employing amultistep dialysis against a solution containing buffer F and adecreasing concentration of urea (from 2 –0 mol/L). The enzymewas purified by size-exclusion chromatography using a HiLoad16/60 Superdex 75 (Amersham Biosciences) and eluted withbuffer G.

Antibody generation and purificationFemale BALB/c mice were immunized with complete Freund's

adjuvant (Disco) and 50 mg of the catalytic domain of LOXL2 andboosted every 2 weeks with incomplete Freund's adjuvant bysubcutaneous injection. Spleens were collected, and B cells werefused with NSO murine myeloma cells. Hybridomas werescreened with ELISA for immunoreactivity against the catalyticdomain of LOXL2, and selected hybridomas positive for LOXL2were subcloned and expanded in tissue culture. Hybridoma cellswere grown in DCCM, and the supernatants were purified on aHiTrap Protein A column equilibrated with 100 mmol/L phos-phate buffer (pH 8) and eluted with 100 mmol/L citrate buffer(pH 6).

ELISA binding assayNinety-six–well plate (Nunc) was coated with LOXL2 or BSA at

5 mg/mL. After blocking with 3% milk in PBS, the plate was

incubated with the antibodies for 1 hour at 25�C. Bound anti-bodies were detected by peroxidase-conjugated antibody goatanti-mouse (Jackson ImmunoResearch). The EC50 was calculatedfrom a 4-parametric sigmoidal curve fitting analysis.

qPCR analysisRNA was isolated from cultured cells using the miRNeasy

Extraction Kit (Qiagen) according to the manufacturer's instruc-tions. cDNA was obtained with cDNA Reverse Transcription Kit(Applied Biosystems). All PCR reactions were performed usingFast SYBR Green PCR Master Mix (Applied Biosystems). Expres-sion levels were calculated by the DCt method after normalizingthe genes with HPRT. The results are presented relative to day 1.Primer sequences are listed in Supplementary Table S2.

Cell lines and cultureThe HDF cell line was a gift from the laboratory of Stephen

Weiss (2012; University of Michigan, Ann Arbor, MI). HDF cellswere cultured in high-glucose DMEM (Invitrogen) supplementedwith 10% (v/v) heat-inactivated FBS (Invitrogen), 100 U/mLpenicillin, and 100 g/mL streptomycin (Biological Industries).The MDA-MB-231–RFP stable cell line was a gift from H. Degani(2014; Weizmann Institute of Science, Rehovot, Israel). MDA-MB-231 cells were grown in RPMI-1640 (Gibco BRL) supplemen-ted with 10% (v/v) heat-inactivated FBS (Invitrogen), 1 mmol/Lsodium pyruvate, 100 U/mL penicillin, and 100 g/mL strepto-mycin (Biological Industries). TheHUEVC cell linewas a gift fromthe laboratory of Gera Neufeld (Technion - Israel Institute ofTechnology, Haifa, Israel; 2012). For all cell lines, the cells wereusedwhen received without further authentication. The cells weremaintained at 37�C in a humidified atmosphere containing 5%CO2, and the medium was exchanged every 2 to 3 days andpassaged after reaching 80% to 90% confluence. For ECM syn-thesis, HDF cells were grown on glass coverslips in 24-well dishesuntil reaching contact inhibition, and the mediumwas replacedand supplemented with 5 ng/mL EGF, 5 mg/mL insulin, and150 mg/mL L-ascorbic acid phosphate magnesium salt n-hydrateto induce ECM secretion, in the presence of vehicle solution(PBS) or GS341 in PBS (100 ng/mL).

ECM isolation from tissue cultureThe HDF cells were incubated with 0.4% Triton, 1.5 mol/L

NaCl, 50mmol/L Tris pH 8, and 50mmol/L EDTA for 48 hours in4�C, washed gently with water, incubated with 0.5% sodiumdeoxycholate for 1 hour at 25�C, and finally with PBS (þMgCl2)and 100 ngr/mL DNase at 37�C for 1 hour to remove DNAcontamination.

ECM isolation from tissueTumor sections (150 mmol/L) were washed three times with

PBS and water, underwent six cycles of freeze and thaw, followedby treatment with NH4OH for 20 minutes.

Two-photon microscopy and second harmonic generationSamples were imaged using 2PM:Zeiss LSM 510 META NLO

microscope equippedwith abroadbandMai Tai-HP-femtosecondsingle box tunable Ti-sapphire oscillator, with automated broad-band wavelength tuning 700 to 1,020 nm from Spectra-Physicsusing a 800-nm wavelength (detection at 400 nm) and a 20�objective.

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Scanning electron microscopyThe ECM was fixed in 4% paraformaldehyde/2.5% glutaralde-

hyde in PBS and incubated with sodium silicotungstate for 40minutes at room temperature. Ethanol dehydration of sampleswas carried out by consecutive washing with increasing concen-tration of ethanol (from 30% to 100%), followed by ethanolexchange with liquid CO2 and critical point drying. Finally,samples were coated with a thin layer of gold and imaged in anultra-microscope (Zeiss).

Image analysisImaging analysis was done by Fiji package (27). Fourier com-

ponent analysis for directionality was performed on data usingthe Fiji plug-in "Directionality" created by Jean-Yves Tinevez(http://pacific.mpi-cbg.de/wiki/index.php/Directionality) andfollowing Fiji's instructions (28).

Mass spectrometryThe proteins were extracted in 8 mol/L urea, 100 mmol/L

ammonium bicarbonate and sonicated. They were then reduced(DTT, 30 minutes, 60�C), modified (iodoacetamide, 30 minutesat room temperature), and digested overnight in 2 mol/L urea,25 mmol/L ammonium bicabonate with modified trypsin (Pro-mega) at a 1:50 enzyme-to-substrate ratio at 37�C. The resultingtryptic peptides were desalted using C18 tips (Harvard) andanalyzedby LC/MS-MSusing aQExactive PlusMass Spectrometer(Thermo Fisher Scientific). The peptide mixture was resolved andmass spectrometry (MS) was performed in a positive mode usingrepetitively full MS scan followed by high collision–induceddissociation of the 10 most dominant ions (>1 charges) selectedfrom the first MS scan.

MS data analysisThe MS data were analyzed using the MaxQuant software

1.5.1.2. (www.maxquant.org) and the Andromeda search engine,searching against the human uniprot database, with mass toler-ance of 20 ppm for the precursor masses and 20 ppm for thefragment ions. Oxidation on methionine proline and lysine andprotein N-terminus acetylation were accepted as variable mod-ifications, and carbamidomethyl on cysteine was accepted as astatic modification. Peptide- and protein-level FDRs were filteredto 1% using the target-decoy strategy. Data were quantified bylabel-free analysis using the same software.

ImmunofluorescenceHDF and MDA-MB-231 cells were grown on coverslips for 48

hours. The cells were then washed, fixed with 4% paraformalde-hyde for 20 minutes, permeabilized using 0.05% Triton X-100,and blocked with 5% BSA in PBS. Staining for LOXL2 wasconducted using rabbit anti-LOXL2 (Santa Cruz Biotechnology)according to the manufacturer's instructions, followed by incu-bation with an anti-rabbit secondary antibody (1:200, AlexaFluor; green). Confocal microscopy was performed as describedpreviously (29). Murine tumors were fixed in 4% paraformalde-hyde, embedded in paraffin, and sectioned. Tumor sectionsunderwent deparaffinization and antigen retrieval according tothe primary antibody manufacturer's instructions. Sections werepermeabilized with 0.5% Triton X-100 in PBS (5 minutes) andblocked with 5% BSA (in PBS containing 0.1% Triton) for 1 hourat room temperature. The samples were incubated overnight at

4�C with a phosphorylated histone H3 antibody (Santa CruzBiotechnology). After three PBS washes, the cells were stainedwith secondary antibodies (Abcam) for 1 hour at room temper-ature followed by 10 minutes of DAPI (4,6-diamidino-2-phe-nylindole dihydrochloride) staining. The cells were viewedunder a Nikon eclipse 90i fluorescence microscope. Pictureswere taken with a 1310 digital camera (DVC). Live cell fluo-rescence microscopy was carried out using the DeltaVisionsystem (Applied Precision). For imaging of focal adhesion,50,000 MDA-MB-231 cells were seeded on decellularized ECMderived from HDF cells in RPMI medium and grown for 72hours. Samples for scanning electron microscopy (SEM) wereprepared as listed above. For immunofluorescence, sampleswere permeabilized using 0.05% Triton X-100 and blockedwith 5% BSA in PBS. Cells were stained for paxillin, phalloidin,and DAPI and visualized by confocal microscopy using aspinning disc microscope.

Cell invasion assaysThe 3D Spheroid Cell Invasion Kit (Trevigen) was used

according to the manufacturer's protocol. Cells (3,000) wereplated in a basement membrane extract and cultured for 72hours. Once spheroids formed, the invasion matrix was addedand antibodies were supplemented in the medium. Imageswere taken after 6 days, and the spheroid area was analyzedwith Fiji software. The quantification of cell invasion was basedon a binary segmentation of phase images of cell spheroidsembedded in the matrix. Each image was segmented andquantified by ImageJ by drawing region of interest around thespheroids and recording the numbers of pixels (correspondingto invasion area) in each image.

Animal experimentsFemale CB-17 SCID mice (Harlan Laboratories; 5 per group)

were implanted in the fat padwith RFP-MDA-MB-231 cells (2.5�106 cells/mouse). After three weeks, mice were treated twice aweek with either the vehicle solution of the antibody (PBS) or 30mg/kg GS341 for a period of four weeks. The tumor volume(width2� length/2)was determined periodically. For lungmetas-tasis, lungs were removed and washed, and images were acquiredusing a fluorescent binocular. Tumor and lung tissues werepreserved in optical cutting temperature and kept frozen at�80�Cuntil used.

Ethical regulationsAnimal experiments were approved by the Weizmann Animal

Care and Use Committee and were executed in accordance withnational guidelines and regulations.

Statistical analysesAll analyses were performed in at least triplicate. Statistical

analysis was performed using GrpahPad Prism 6.

ResultsScreening and identification of mAb clones capable ofinterfering with fiber assembly

To characterize the effect of crosslinking modulators on ECMassembly and organization in a physiologically relevant micro-environment, we searched for a cell line that synthesizes a nativeECM scaffold by expressing various ECM components, as well as

Disruption of Collagen Fibril Alignment Attenuates Cancer

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all members of the LOX family. Several cell lines are known toexpress LOX enzymes, including HDF, HUVECs, MCF-7, andMDA-MB-231, and were evaluated for their ability to producefibrillary ECM components that can be visualized by secondharmonic generation (SHG) and SEM. The 3D matrix depositedby the HDF cell line was composed of a variety of ECM compo-nents, including fibrillary proteins, and appeared to mimic thephysiologic conditions of the actualmicroenvironment createdbymesenchymal stroma (30) and was selected for further charac-terization. We grew the activated fibroblasts on a glass coverslipfor a period of 7 days and monitored the synthesis and assemblyof fibrillary collagen in its native, unlabeled state by two-photonSHG microscopy (Fig. 1A; ref. 31). After 5 days of growth, westarted to detect a SHG signal, indicating synthesis of a premature,disorganized form of collagen (Fig. 1B), and two days later, weobserved the alignment of collagen fibers in a specific direction,demonstrating the level of complexity required to construct the

ECM. As determined by real-time PCR analysis, HDF cells expressall members of the LOX family (Fig. 1C). The results show thatexpression of these enzymes is dynamically regulated during ECMsynthesis, indicating the distinct roles they possibly play duringECM build-up.

We generated anti-LOXL2–specific antibodies that target theactive site, in contrast to the previously reported anti-LOXL2antibody that targets noncatalytic regions of LOXL2 (21). Fol-lowing hybridoma library generation, we obtained several mAbclones displaying binding affinities to the catalytic domain ofLOXL2 at the subnanomolar range (Supplementary Fig. S1).These mAbs were added to the medium of the HDF cells at day0. After 7 days, we screened, using SHG, for an antibody thatproduces any sort of visible variation in fibrillary collagen mor-phology. The irreversible pan inhibitor of LOX enzymes, b-ami-nopropionitrile (bAPN), served as a control for collagen synthesisalterations (32). Fibrillary proteins synthesized by cells treated

Figure 1.

Identification of LOXL2 antibody interfering with ECM assembly. A, scheme illustrating the experimental setup for monitoring different stages in collagenassembly secreted by HDF cells using SHG microscopy. B, representative SHG (top) and bright-field (bottom) images of fibrillary collagen synthesized by HDFcells at various time points. C, qPCR analysis of gene-encoding members of the LOX family. Displayed are the average values � SEM of three independentbiologic replicates. D, schematic illustration of the screen assay. Mice were immunized with the catalytic domain (orange, residues 545–775) of the LOXL2 gene.Positive clones (active) were considered to be antibodies that caused misaligned collagen morphologies when supplemented to the HDF cells medium. E,representative SHG images of the screen results for the control polyclonal IgG antibody, bAPN, GS341, and GS092, which highlight GS341 as a potentantibody that interferes with ECM alignment. Representative fiber directionality analysis plots are overlaid on each SHG image depicting the frequency offibers in a specific orientation.

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with several anti-LOXL2 antibodies (GS092 shown as an exam-ple) after a week-long incubation exhibited the typical linear andaligned collagen organization observed in the IgG control–treatedcells (Fig. 1E). Upon treatment with bAPN,we could hardly detectany aligned fibers in the matrix, and the ECM spontaneouslyruptured during manual handling of the sample. In contrast,treatment with the antibody clone designated GS341 did lead tothe formation of collagen fibers, but they were oriented inmultiple directions as demonstrated by SHG (Fig. 1E).

To quantify the changes in directional fiber orientation, weapplied Fourier component analysis for directionality on rep-resentative images obtained by SHG. Strikingly, while thedirectionality histogram characteristic of fibrillary proteinsfrom HDF cell samples treated with control IgG or othernonactive antibody clones (such as GS092) portrayed a singlepeak at a preferred orientation, the histogram of fibrillaryproteins from HDF cell samples treated with GS341 containedmultiple peaks (Fig. 1E and Supplementary Fig. S2), indicatinga completely isotropic behavior. We, therefore, considered thisantibody a potential ECM-assembly modulator and selected itfor further characterization.

We evaluated the binding affinity and inhibitory activity ofGS341, in comparisonwith the clone that didnot induce apparentchanges in fibril collagen morphology, GS092. Although thebinding affinities (EC50) of the two clones to the catalytic domainof LOXL2were both in the sub-nanomolar range (SupplementaryFig. S1), the dissociation constant (KD) of GS341 (27 nmol/L),determined by microscale thermophoresis, was 5-fold lowercompared with GS092 (122 nmol/L; Supplementary Fig. S3). Inaddition, we examined the inhibitory activities of the two cloneson the crosslinking activity of LOXL2 using collagen type I.Importantly, the inhibitory crosslinking activity of GS341 onLOXL2 was more pronounced than that of GS092 (Supplemen-tary Fig. S4). Thus, the antibodies are different in inhibitorycrosslinking activities and in the dissociation constant, whichmay rationalize why treatment of HDF cells with GS341 and notGS092 leads to disorganized collagen fibers, due to strongerinhibition of collagen crosslinking.

To gain structural insights into the bindingmode of GS341, weconducted competition experiment between the antibody andelastin, one of the main substrates of the LOX enzymes. Titrationof preincubated complex of LOXL2 and GS341 with increasingconcentration of elastin did not induce any changes in thermo-phoresis (see Supplementary Materials and Methods), suggest-ing that the substrate cannot bind to LOXL2 when it is bound toGS341 (Supplementary Fig. S3C). Although further structuralinvestigation is required to define the exact epitope that GS341recognize, due to the lack of available crystal structures of LOXs,we canonly estimate thatGS341 interfereswith substrate binding.Thus, GS341 prevents the assembly of linear collagen fibers, asdemonstrated by SHG, by directly and specifically inhibitingcollagen crosslinking mediated by LOXL2, possibly by interferingwith substrate binding.

Western blotting of lysates and supernatants of fibroblastsusing the GS341 antibody did not identify any cross-reactivityof the antibody with other family members (Supplementary Fig.S5). In contrast, Western blotting of mouse LOXL2 did revealcross-reactivity of the antibody with both human and mouseLOXL2 (Supplementary Fig. S5), which further enabled the char-acterization of the inhibitory effect of the antibody both in vitroand in vivo.

Anti-LOXL2 antibody alters the orientation and thickness, butnot the composition, of fibrillary ECM proteins

SEM investigation of the native and treated ECM scaffoldsprovided us with nanoscale imaging not only of fibrillarycollagens but also of other protein assemblies within the ECM(Fig. 2A). The native ECM secreted by the control HDF cellscontained mainly fibrillary proteins with D-banding periodic-ity, most of which were aligned along the fiber axis (Fig. 2A andC). Treatment with GS341 disrupted this alignment; while thefibrils in the control samples assembled in one dominantdirection, those in the antibody-treated samples were dispersedand orientated in a multitude of directions, as gathered fromthe representative directionality analysis (Fig. 2B and C) dis-playing anisotropic ECM fibril organization. Reduction in theaverage width of fibrils within the fibers treated with GS341further indicated that inhibition of LOXL2 also caused internalfibril crosslinking defects (Fig. 2D). As the full repertoire ofLOXL2 substrates is unknown, we cannot claim that theobserved changes occur only in fibrillary proteins, althoughthe evidence collected from the multiple imaging methodsapplied strongly indicates that collagens are the proteins mostaffected.

MS and bioinformatics analyses further quantified the effect oftreatment with the GS341 antibody on the entire ensemble ofECMproteins.Our initial, unbiasedMSanalysis ofwhole proteinsextracted from the HDF-derived matrix with or without theantibody identified2,865proteins, among them111ECM-relatedproteins and enzymes (Fig. 2E andSupplementary Fig. S6; ref. 33).Strikingly, none of these proteins displayed any significant dif-ference in abundance following treatment with the antibody(t test, P < 0.05), implying that the inhibition of LOXL2 onlyaffects the topography of the ECM, not its overall composition.This is in agreement with the recent observation that bAPNinhibition of LOX expressed by tendon fibroblasts does not altergene expression or the content of key ECMproteins; it only affectsthe physical properties of the collagen fibrils (34). Overall, inhi-bition of LOXL2 byGS341 prevents proper alignment of fibrillaryproteins, such as collagen, without affecting ECM composition.

Anisotropic ECM fibril organization disrupts cancer cellproliferation, adhesion, invasion, and migration

ECM composition and geometry affects epithelial tumor cellproperties, such as growth, differentiation, and adhesion (30).Particularly in breast tumors, the formation of a dense meshworkof primarily crosslinked collagen fibers has been shown to triggerEMT and cell invasion (35), to facilitate intravasation and distantmetastasis formation (36). We, therefore, examined the influenceof GS341-treated matrices on human breast cancer cell behavior.In the tumor microenvironment, LOXL2 is not expressed only byresident fibroblasts but also by aggressive cancer cells, such as thetriple-negative breast cancer MDA-MB-231 cells (37), as demon-strated by blotting and immunostaining for LOXL2 (Supplemen-tary Fig. S7). This cell linewas chosenas a representative cell line toexamine the effect of altered matrix topography on cancer cellbehavior.

The native matrix scaffolds synthesized by HDF cells werecarefully decellularized, and MDA-MB-231 cells were seeded ontop of it andmonitored for 72 hours, the time period required forfull adhesion to and invasion into the matrix (Fig. 3A). Immu-nofluorescence analysis of the cancer cells for paxillin, amarker formature focal adhesion sites, revealed an alteration in cell adhesion






to the LOXL2-deficient ECM (Fig. 3B), which complemented thechanges in cellmorphologydetectedby SEM imaging.Cancer cellsdisplayed an elongated morphology when cultured on nativeECM, whereas cancer cells grown on ECMderived from fibroblastcells treated with GS341 exhibited a rounded morphology withfewer extensions to the ECM (Fig. 3C), as can also be gatheredfrom the notable reduction in the width-to-length aspect ratios ofcell bodies (Fig. 3D).

To assess the effect of the disrupted matrix alignmentconferred by GS341 on cell migration, we seeded fluorescentlylabeled MDA-MB-231 cells on ECM derived from fibroblaststreated either with vehicle or GS341 and followed their move-ment characteristics using live cell imaging. Unlike the randommovement of MDA-MB-231 cells often detected on 3D Matri-gel (38), we observed a slow but persistent movement of thecells aligned to the fibers' axis. Cell motility was significantlyfaster on the disorientated and thinner collagen fibers derivedfrom fibroblasts treated with GS341, with an average velocityof 0.05 � 0.01 mm/minute (Supplementary Movie 2) com-pared with that of 0.03 � 0.05 mm/minute in the control

samples (Supplementary Movie 1; Fig. 3E). It was recentlydemonstrated that collagen alignment enhances the efficiencyof directional persistence and promotes protrusions alongaligned fibers (39), movement that is somewhat slower com-pared with random cell movement. The increase in cell veloc-ity on the misaligned collagen fibers could be rationalized bythe lack of directional persistency of the cells during move-ment due to the randomness of fiber orientation post treat-ment with GS341, as demonstrated by quantification of theorientation of fibrillary protein in the matrices (Fig. 2E). Thus,misaligned fibers may contribute to increase in cell velocity bydecreasing the cells' directional persistency. Although othercancer cell lines may exhibit differential cell behaviors, ourresults provide evidence for the manner by which fiber mis-alignment, due to LOXL2 inhibition, has the ability to inter-fere with cellular function.

To examine the direct individual effect of inhibiting the activityof LOXL2 secreted by the cancer cells on ECM remodeling,independently from the effect of the matrix itself, we evaluatedwhether GS341 inhibits the tumor cells' ability to penetrate

Figure 2.

Anti-LOXL2 disrupts the orientation and thickness of fibrillary ECM proteins secreted by fibroblasts. A and B, SEM analysis of collagen fibril morphology upontreatment of HDF cells with GS341 reveals differences in fiber alignment and width. Shown are representative images of decellularized ECM samples at twodifferent magnifications. C, fibril directionality analysis done by Fiji on the lower magnification representative images. D, fibril diameter measurements reveal areduction in fibril average diameter due to treatment with GS341 (statistics are obtained from three independent biologic replicates using one-sided t test;the number of measured fibrils for diameter quantification was at least n¼ 80). E, global proteomic MS analyses of fibroblast cells and the synthesized ECM did notidentify a statistically significant difference in total protein abundance upon treatments with GS341 (results obtained from quadruplets). Scale bar, relativeprotein abundance. F and G, magnification of the MS results regarding collagens and representative ECM-associated proteins in the samples.

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commercial Matrigel or collagen-based barriers. Interestingly,although invasion through Matrigel matrices was not affected(Supplementary Fig. S8A), a dose-dependent inhibition of inva-sion in a 3D assay containing collagen type I was observed (Fig. 3Fand H). In contrast, GS092 antibody that was shown not tointerfere with collagen fibril alignment did not affect the invasioncapability ofMDA-MB-231 (Supplementary Fig. S8B). SHG imag-ing at the spheroid border showed a reduction in the fibrillarycollagen signal following treatment with GS341 (Fig. 3G and I),suggesting that the activity of LOXL2 secreted by the cancer cells iscritical for mediating matrix remodeling during invasion. Thus,inhibition of LOXL2 not only impacts cellular processes depen-dent on fibrillary collagen alignment but may also be responsiblefor attenuating cancer invasion processes mediated by LOXL2-dependent local collagen remodeling.

Finally, since LOXL2 is also implicated as a modulator ofangiogenesis (25), we tested the effect of the antibodies in a tubeformation assay. GS341 strongly inhibited the spontaneous for-mation of tubes by HUVECs, whereas GS092 did not have asignificant effect on this process (Supplementary Fig. S9). Thisresult suggests that GS341 may also contribute to inhibition offunctional properties of other cell lines, such as HUVECs, whichare also known to play key roles in tumor invasion andmetastasis,in ECM-dependent and independent manners.

Normalization of ECM fibril organization at the tumormicroenvironment attenuates cancer growth

To test in vivo whether interference with fibrillary protein align-ment could inhibit breast cancer progression, we injected MDA-MB-231 cells into mammary fat pads of immunocompromised

Figure 3.

Disoriented ECM fibril organization disrupts cell invasion, adhesion, and migration. A, scheme illustrating the experimental setup for studying the effect of matrixproperties on cancer cell behavior. MDA-MB-231 cells were seeded on decellularized ECMs synthesized by the HDF cells, and their adhesion, morphology, andmigratory properties were assessed by different modalities. B, immunofluorescence staining for actin and paxillin in MDA-MB-231 cells seeded on ECM derivedfrom control and GS341-treated HDF cells. C, representative SEM images of MDA-MB-231 cells seeded on ECM derived from control and GS341-treatedHDF cells showing a change in cancer cell morphology. D, aspect ratios of MDA-MB-231 cell bodies demonstrate a reductionwhen seeded on GS341-treatedmatrices(statistics were obtained from three independent biologic replicates using one-sided t test; the number of measured cells was at least n ¼ 10). E, cell velocityon the misaligned ECM derived from the GS341-treated cells was increased compared with movement of native ECM. F and H, GS341 inhibits the invasionof MDA-MB-231 cells in a dose-dependent manner in a 3D spheroid invasion assay. G and I, SHG imaging at the spheroid border demonstrates a reduction incollagen signal intensity upon treatment with GS341. Displayed are the average values � SEM.






SCID mice. As genetic knockdown of LOXL2 in a similar breastcancer model using MDA-MB-231 cells indicated that LOXL2 isnot required for the initiation of tumor growth (18), we startedtreatmentwithGS341 threeweeks after tumor establishment for aperiod of four weeks. Treatment with GS341 led to a significantdecrease in the final tumor volume (Fig. 4A) and also affected, at alowermagnitude, the number of lungmetastases (SupplementaryFig. S10). The treatment significantly reduced cell proliferation inthe primary tumors, as shown by immunostaining analysis for amarker of cell-cycle activity (Fig. 4B). The tumors treated withGS341 were more fragile, as shown by H&E staining of tumorsections, and contained statistically significantly lower amountsof crosslinked collagen, as depicted by Sirius red staining (Fig.4C), indicating direct inhibition of fiber crosslinking by inhibi-tion of LOXL2.

To confirm that the decrease in tumor growth is directly linkedto GS341's modulation of collagen crosslinking, we quantifiedthe changes in fiber topography of the primary tumor. Our SEM

analysis (Fig. 4D) of decellularized tumor sections found thefibrillary proteins in the GS341-treated tumors to have a differentmorphology than the control tumors. The fibrils in the controltumor sections were aligned in one dominant direction, whereasthe fibrils in tumor sections obtained from mice treated withGS341wereoriented inmultiple directions. In addition, thefibrilsin the GS341-treated tumors varied in their diameter and werethinner in comparison with the vehicle-treated tumors (Fig. 4Eand F), recapitulating the effect onmatrix topography observed inthe in vitro fibroblast experiment (Fig. 2). These results emphasizethat the in vitro screening setup for collagen fibril modulatorsindeed resembles the ECM topography detected in vivo. In addi-tion, disoriented collagen fibers in the tumor microenvironmentcan also prevent invasion of disseminating breast cancer cells,which preferentially move along bundled collagen fibrils (40).Thus, in vivo inhibition of fibril crosslinking at the late stages ofECM assembly, as was brought about by our anti-LOXL2 mAb,represents an effective means to control the formation of the

Figure 4.

Perturbation of collagen fiber linearization decreases tumor growth. A, quantification of tumor volume of vehicle or GS341 (30 mg/kg, twice weekly)-treatedmice. Displayed are the average values � SEM, at least n ¼ 5 mice per group. B, immunofluorescence staining of paraffin tumor sections for phosphohistoneh3. Statistics were obtained from 3 mice per group; the number of cells quantified was at least n ¼ 10. C, hematoxylin and eosin (H&E) staining of tumorparaffin sections shows relaxation of the tumor scaffold following treatment with GS341. Sirius red staining of tumor sections depicts a reductionin crosslinked collagen upon treatment with GS341. Right, quantitative analysis. D–F, SEM analysis of tumor collagen fibril morphology and quantificationof fibril directionality (statistics were obtained from 3 mice per group; the number of measured fibrils for diameter quantification was at least n ¼ 80).

Grossman et al.





aligned fiber arrangement important for tumor growth andinvasion.

DiscussionCellular behavior and proper tissue function are largely depen-

dent on and regulated by protein fibers, such as collagen, withinthe ECM. ECMhomeostasis, which is critical for organ function, ismaintained through a delicate balance between ECM proteinsynthesis, protein fibril assembly and polarization, posttransla-tional modification, remodeling, and degradation (41). Disrupt-ing this balance results in various pathologic conditions,highlighting the correlation between altered ECM structural orbiophysical properties and disease grade and subtype (42–44).Tissue engineering studies have demonstrated that changes in anyof the physical properties or components of the ECM can affectcellular behavior. It has been recently shown that MDA-MB-231cell migration is dependent on collagen type I fibril alignmentrather than stiffness (39, 45), and that collagen fibril diameterregulates cell morphology, invasiveness, and cluster formation(46). This understanding has made the development of strategiesaimed at normalizing tissue ECM assembly and remodelingattractive for clinical use.

Our system was found to be ideal to screen for modulators offibrillary collagen assemblies in an efficient and robust way,without the need for complicated sample preparation procedures.Remarkably, the effect on collagen fibril topography detected bySEM in the 3Dmatrix system was recapitulated in the experimen-tal in vivo test of the selected antibody, highlighting the clinicalrelevance of it. Therefore, controlling disease progression byprotecting the microenvironment by maintaining normal fibrilorientation and thickness during remodeling can serve as a newfrontier inmedicine aimed at attenuating ECM-associated pathol-ogies. This approach can serve for screening inhibitors of variousECM remodeling enzymes impacting different biophysical prop-erties of the ECMscaffold. In addition, it provides an experimentalset up to further screen and analyze the impact of native collagenalignment on different cancer cells.

In addition, we revealed newmolecular insights into the role ofLOXL2 in collagen assembly inpathologic scenarios. Inhibition ofLOXL2 resulted in selective attenuation of the supramolecularcollagen assembly within tumorigenic cellular microenviron-ment. Through its crosslinking activities, LOXL2 is responsible

for the supramolecular bundling of aligned collagen fibrils in theECM. Specifically, LOXL2 controls thenano- andmicroscale levelsof collagen assembly by affecting both final fiber bundle width(nanoscale) and fiber orientation (microscale). Our findingsreveal that collagen fibril alignment mediated by LOXL2 directlycontrols cancer-associated cellular processes driven by the orien-tation and directionality of bundled collagen fibers. Thus, themisalignment of collagen fibers due to inhibition of LOXL2suggests that LOXL2 controls fibril alignment so as to tune ECMtopography.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. Grossman, N. Ben-Chetrit, Y. Yarden, I. SagiDevelopment of methodology: M. Grossman, N. Ben-Chetrit, I. Solomonov,I. SagiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):M. Grossman, N. Ben-Chetrit, A. Zhuravlev, E. BassatAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Grossman, N. Ben-Chetrit, A. Zhuravlev, R. Afik,E. Bassat, I. SolomonovWriting, review, and/or revision of the manuscript: M. Grossman, N. Ben-Chetrit, A. Zhuravlev, I. Solomonov, I. SagiStudy supervision: I. Sagi

AcknowledgmentsThe authors thank Dr. Leitner, Hedva Hamawi, Anna Aloshin, Dr. Zaffryar-

Eilot, Dr. Kalchenko, and Dr. Kartvelishvily for their helpful technical assis-tance, and Dr. Miriam and Sheldon G. Adelson Medical Research Founda-tion and the Rising Tide Foundation.

Grant SupportY. Yarden is supported by the European Research Council and the Israel

Cancer Research Fund. I. Sagi is supported by the Israel Science Foundation, theMSKCC-Weizmann fund, the Thompson foundation, and the Ambach FamilyFund.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 14, 2015; revised March 30, 2016; accepted May 2, 2016;published OnlineFirst May 24, 2016.

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Grossman et al.




2016;76:4249-4258. Published OnlineFirst May 24, 2016.Cancer Res Moran Grossman, Nir Ben-Chetrit, Alina Zhuravlev, et al. Fibril Alignment That Control Assembly of the Extracellular MatrixTumor Cell Invasion Can Be Blocked by Modulators of Collagen

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