100 | CANCER DISCOVERY JULY 2011  www.aacrjournals.org

The first Line of intra-abdominal Metastatic Attack: Breaching the Mesothelial Cell Layer Hilary A. Kenny, Kristin M. Nieman, Anirban K. Mitra, and Ernst Lengyel

summary: Iwanicki and colleagues reveal that ovarian cancer spheroids clear mesothelial cells which cover the surface of the abdominal cavity using myosin-generated force. Cancer Discovery; 1(2); 100–2. ©2011 AACR .

in THe sPOTLiGHT

model systemsand intraperitoneal imagingtechniques thatallowinvestigationoftheseearlyevents.Mesothelialcellsarethoughttobethefirstlineofdefenseagainstallabdominallymetastasizingtumors.Theinitialeffectsofmesothelialcellson ovarian cancer cell adhesion and invasion have been ex-ploredin vitro.In1985,Niedbalaandcolleagues(4)presentedan in vitro model system co-culturing primary human me-sothelial cells grown on extracellular matrix-coated culturedisheswithprimaryovariancancertumorcellsfromascites.This study found that ovarian cancer cells attach more ef-ficiently to extracellular matrix as compared to mesothelialcells. A subsequent study used primary human peritonealmesothelial cells and fibroblasts with extracellular matrix,and discovered that mesothelial cells inhibit ovarian cancercell adhesion and invasion, whereas fibroblasts had the op-positeeffect(5).Theinhibitoryeffectofmesothelialcellsonovarian cancer cell adhesion dissipated with the senescenceof mesothelial cells (6). These studies established a role formesothelial cells as the “first line of defense” against intra-abdominal cancer cell metastasis. However, the mechanismbywhichcancercellsclearmesothelialcellstogainaccesstothebasementmembraneremainedelusive.

Iwanicki and colleagues (7) developed and employed alive,real-timeimage-basedin vitromodeltogainspatialandtemporalresolutionofthe initial interactionbetweenovar-iancancertumorspheroidsandmesothelialcells.Throughthe use of cutting-edge time-lapse fluorescent microscopy,they monitor and measure the interaction of ovarian can-cer cells and green fluorescent protein–labeled mesothelialcells,includingmesothelialcellclearance.Theirveryelegantstudy appears in the current issue of Cancer Discovery andprovides novel insights into the initial interaction betweencancercellsandmesothelialcells.Thekeymechanismeluci-datedbytheauthorsisovariancancerspheroidsusemyosin-generatedforceto“breach”andremovethemesothelialcellmonolayer, termed “mesothelial cell clearance” (illustratedinFig.7ofref.7).

Cellsexertforceontheirenvironmentthroughassociationofmyosinand integrinnetworksusingrecruitmentof talinI to adhesion sites (8). The authors establish that attenua-tionofmyosin II,usingacombinationofmyosin IIAshorthairpinRNA(shRNA)andmyosinIIBshortinterferingRNA(siRNA)treatment, inhibitstheabilityoftheovariancancercells to clear mesothelial cells. However, decreasing myosinII has no effect on mesothelial cell apoptosis as measured

In 2010, more than 21,000 women were diagnosed withovariancancerintheUnitedStates.Themajorityofpatientspresent with advanced-stage disease, after tumors have me-tastasizedthroughouttheabdominalcavity.Despiteaggres-sive surgery and chemotherapy, less than 30% of patientswith widely spread intra-abdominal ovarian cancer attainlong-termprogression-freesurvival (1).Thebiologyofovar-ian cancer differs from hematogenously spreading tumors,such as breast and colon, in that the cells detach from theprimaryovariantumoranddisseminatethroughouttheperi-toneal cavity by the clockwise flow of peritoneal fluid (2).Successful implantation is characterized by the adhesion,migration,proliferation,andinvasionofthetumorcellsintothe peritoneal surface or the omentum. The peritoneal cav-ity is also a site of metastasis for other epithelial cancers,includinggastric,colon,appendiceal,andpancreaticcancer,which have metastatic patterns that parallel ovarian cancerdissemination.Patientsdiagnosedwiththesecancersgener-ally present at a late stage, thus micrometastases are rarelyevident.Thesecircumstancescreateaneminentchallengetoinvestigatetheinitialinteractionbetweencancercellsandtheperitonealsurface.Weare“inthedark”regardingourknowl-edgeofthefirststepsofmetastasisintheabdominalcavity.

Theperitonealcavityislinedbyacontinuoussinglelayerof mesothelial cells (Fig. 1A), a unique cell type that coversonlyperitoneal,pleural, andpericardial serosal surfaces (3).However, mesothelial cells are absent under the peritonealtumormass inwidespreaddisease(Fig.1B).Anexplanationfor the absence of mesothelial cells in advanced disease isuncertain.Ithasbeensuggestedthatthefragilemesothelialcells easily detach from the peritoneal surface, making thecollectionofandobservationof theirearly interactionwithtumorcellsevenmoredifficult.

Contributingtothemysterysurroundingtumorandme-sothelialcellinteractionisthegenerallackofadequatein vivo

Authors’ Affiliation: Department of Obstetrics and Gynecology/Section of Gynecologic Oncology – Center for Integrative Science, University of Chicago, Chicago, Illinois

Commentary on Iwanicki et al., p. 144(7).

Corresponding Author: Ernst Lengyel, University of Chicago, Department of Obstetrics and Gynecology/Section of Gynecologic Oncology, 5841 South Maryland Avenue, Chicago, IL 60637. Phone: 773-702-6722; Fax: 773-702-5411; E-mail: elengyel@uchicago.edu

©2011 American Association for Cancer Research.doi: 10.1158/2159-8290.CD-11-0117

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byimmunoblotandimmunofluorescentanalysisofcleaved-caspase3.Iwanickiandcolleagues(7)discoverthatblockingtalin I expression in cancer spheroids by treatment with ta-linIshRNAinhibitsovariancancer–inducedmesothelialcellclearance. The authors then report that talin I recruitmentis required for myosin-generated mesothelial cell clearance.Blockingα5β1-integrininovariancancercellsexpressinghighlevels of α5β1-integrin decreases mesothelial cell clearance,whileoverexpressingα5β1-integrin increasesmesothelialcellclearance.Takentogether,thesedatarevealthattheovariancancerspheroidclearsthemesothelialcells fromitspathinanα5β1-integrin-andtalin-dependentmanner.

Alternativemechanismsofmesothelialclearancehavebeenpreviously explored in vitro. Scanning electron microscopyshowedthatovariancancercellsdisruptedintracellularjunc-tions, leading to the retraction of mesothelial cells and theexposureoftheunderlyingextracellularmatrix(4).Further,cancercellsmayclearmesothelialcellsbyinducingapoptosisin the mesothelial cells. Treatment of a colonic cancer cellline with an inhibitory Fas-ligand antibody reduced meso-thelial cell apoptosis, suggesting that tumor-induced meso-thelialcellapoptosisismediated,inpart,byaFas-dependentmechanism(9).Similarly,Iwanickiandcolleagues(7)foundthat ovarian cancer spheroids clear mesothelial cells at thesiteofcontact;however,theovariancancercellshavenoef-fectonmesothelialcellapoptosis.

SupportingthefindingsbyIwanickiandcolleagues(7) isa study, using Fourier transform traction microscopy, thatdescribed a role for the fibronectin receptor, α5β1-integrin,in myosin-generated force (10). Cancer cell lines from vari-ous organs expressing high levels of α5β1-integrin showedincreased invasion through dense 3D collagen fiber matri-ces and were able to generate very strong contractile forceswhencomparedtocellsexpressinglowlevelsofα5β1-integrin(10). Accordingly, treatment of ovarian cancer cells withα5β1-integrin–specific antibody or siRNA significantly in-hibited in vivo attachment, metastasis, and even survivalof mice injected intraperitoneally with ovarian cancer celllines(11, 12).Takentogether,thesestudiessuggestthattheα5β1-integrin–driven“mechanicalforce”ofcancercellsisin-strumentalintheclearanceofmesothelialcellsandtheinva-sionoftheextracellularmatrix(7,10,11).

The tumor spheroid-induced integrin-talin-myosin forcethat was able to clear mesothelial cells was studied by theauthors using a benign immortalized mesothelial cell line(LP-9)andlaterpassagesofprimaryhumanmesothelialcells.Acaveattothisapproachistherecentfindingthatthemor-phologyandsenescentstateofearlypassagesofprimaryhu-man peritoneal cells is different than late-passage primarymesothelial cells (6). The authors reported a difference inthe tumor-induced migratory response of mesothelial cellsfrom the pleural and peritoneal cavities; thus the source of

A

B

Figure 1. Ovarian cancer cell metastasis. Hematoxylin and eosin-stained human peritoneal surface from a cancer-free patient (A) and a microscopic tumor implant on the omental surface from a patient with metastatic serous ovarian carcinoma (B). Mesothelial cells (arrows) line the abdominal surface but are not detected under the proliferating ovarian cancer cell implant (asterisk).

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3. Zhang X, Pettengell R, Nasiri N, Kalia V, Dalgleish A, Barton D.Characteristicsandgrowthpatternsofhumanperitonealmesothe-lialcells:comparisonbetweenadvancedepithelialovariancancerandnon-ovariancancersources.JSocGynecolInvestig1999;6:333–40.

4. NiedbalaMJ,CrickardK,BernackiR.Interactionsofhumanovariantumorcellswithhumanmesothelialcellsgrownonextracellularma-trix.Aninvitromodelsystemforstudyingtumorcelladhesionandinvasion.ExpCellRes1985;160:499–513.

5. KennyHA,KrauszT,YamadaSD,LengyelE.Useofanovel3Dcul-turemodeltoelucidatetheroleofmesothelialcells,fibroblastsandextra-cellular matrices on adhesion and invasion of ovarian cancercells.IntJCancer2007;121:1463–72.

6. Kiazek K, Mikula-Pietrasik J, Korybalska K, Dworacki G, Jörres A,Witowski J.Senescentperitonealmesothelial cellspromoteovariancancercelladhesions.AmJPathol2009;174:1230–40.

7. IwanickiM,DavidowitzRA,NgMR,BesserA,MuranenT,MerrittM, et al. Ovarian cancer spheroids use myosin-generated force toclearthemesothelium.CancerDiscovery2011;1:144–57.

8. Bershadsky AD, Balaban NQ, Geiger B. Adhesion-dependent cellmechanosensitivity.AnnuRevCellDevBiol2003;19:677–95.

9. HeathR,JayneD,O’LearyR,MorrisonE,GuillouP.Tumor-inducedapoptosisinhumanmesothelialcells:amechanismofperitonealin-vasionbyFasLigand/Fasinteraction.BrJCancer2004;90:1437–42.

10. MierkeCJ,FreyB,FellnerM,HerrmannM,FadenL.Integrina5b1facilitatescancercellinvasionthroughenhancedcontractileforces.JCellSci2011;124:369–83.

11. MitraAK,SawadaK,TiwariP,MuiK,GwinK,LengyelE.Ligandin-dependentactivationofc-Metbyfibronectina5b1-integrinregulatesovariancancerinvasionandmetastasis.Oncogene2011;30:1566–76.

12. YokoyamaY,SedgewickG,RamakrishnanS.Endostatinbindingtoovarian cancer cells inhibits peritoneal attachment and dissemina-tion.CancerRes2007;67:10813–22.

mesothelial cells likely has an effect on the interaction oftumorandmesothelialcells.Lastbutnotleast,theperitonealmicroenvironment is not composed exclusively of mesothe-lial cells but is a complex microenvironment of mesothelialcells, fibroblasts, inflammatory cells, and extracellular ma-trices (i.e., fibronectin, vitronectin, and collagen type I) af-fecting the interaction of tumor and mesothelial cells (2).Additional investigationsusing thisnew imagingtechniquewillallowustofurtherelucidateearlyabdominalmetastasisandtesttheeffectsofdifferenttherapiesthatwillbeusefulforadjuvanttherapyaftercompletetumorremoval.

In summary, Iwanicki and colleagues (7) provide the firstevidencethatovariancancerspheroids“force”themesothelialcells out of their way, leading to mesothelial clearance. Thisdata,togetherwiththatfromotherstudies(4,5),suggestthatthecancercells“plow”throughtheprotectivelayerofmeso-thelialcells.Oncethecancercellsreachtheextracellularma-trix,theyhaveaccesstounderlyingstromalcells,whichcanberecruitedtosupporttheirsurvivalandrapidgrowth.

Disclosure of Potential Conflicts of interestNopotentialconflictsofinterestweredisclosed.

PublishedonlineJuly18,2011.

RefeRenCes 1. JemalA,SiegelR,XuJ,WardE.Cancerstatistics,2010.CACancerJ

2. LengyelE.Ovariancancerdevelopmentandmetastasis.AmJPathol2010;177:1053–64.

Clin201 ;60:277–300.0

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