the first line of intra-abdominal metastatic attack...

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

views

on August 23, 2019. © 2011 American Association for Cancer Research.cancerdiscovery.aacrjournals.org Downloaded from

JULY 2011�CANCER DISCOVERY | 101

views

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).

on August 23, 2019. © 2011 American Association for Cancer Research.cancerdiscovery.aacrjournals.org Downloaded from

102 | CANCER DISCOVERY JULY 2011  www.aacrjournals.org

views

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

on August 23, 2019. © 2011 American Association for Cancer Research.cancerdiscovery.aacrjournals.org Downloaded from

2011;1:100-102. Cancer Discovery   Hilary A. Kenny, Kristin M. Nieman, Anirban K. Mitra, et al.   Mesothelial Cell LayerThe First Line of Intra-abdominal Metastatic Attack: Breaching the

  Updated version

  http://cancerdiscovery.aacrjournals.org/content/1/2/100

Access the most recent version of this article at:

  Cited articles

  http://cancerdiscovery.aacrjournals.org/content/1/2/100.full#ref-list-1

This article cites 12 articles, 3 of which you can access for free at:

  Citing articles

  http://cancerdiscovery.aacrjournals.org/content/1/2/100.full#related-urls

This article has been cited by 6 HighWire-hosted articles. Access the articles at:

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgat

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerdiscovery.aacrjournals.org/content/1/2/100To request permission to re-use all or part of this article, use this link

on August 23, 2019. © 2011 American Association for Cancer Research.cancerdiscovery.aacrjournals.org Downloaded from