Muinonen-Martin, AJ et al. (2014) Melanoma cells break down LPA to establish local gradients that

18 Pages·2014·7.41 MB·English

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Muinonen-Martin, A. J. et al. (2014) Melanoma cells break down LPA to
establish local gradients that drive chemotactic dispersal. PLoS Biology, 12
(10). e1001966. ISSN 1544-9173

Copyright © 2014 The Authors

http://eprints.gla.ac.uk/101796

Deposited on: 27 January 2015

Enlighten – Research publications by members of the University of Glasgow
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Melanoma Cells Break Down LPA to Establish Local
Gradients That Drive Chemotactic Dispersal
AndrewJ.Muinonen-Martin1,2,3,OliviaSusanto1,QifengZhang4,ElizabethSmethurst4,WilliamJ.Faller1,
Douwe M. Veltman1, Gabriela Kalna1, Colin Lindsay1,5, Dorothy C. Bennett6, Owen J. Sansom1,
Robert Herd7, Robert Jones1,5, Laura M. Machesky1, Michael J. O. Wakelam4, David A. Knecht8,
Robert H. Insall1*
1CRUKBeatsonInstitute,Glasgow,UnitedKingdom,2YorkTeachingHospitalNHSFoundationTrust,York,UnitedKingdom,3TheLeedsTeachingHospitalsNHSTrust,
Leeds,UnitedKingdom,4TheBabrahamInstitute,Cambridge,UnitedKingdom,5BeatsonWestofScotlandCancerCentre,Glasgow,UnitedKingdom,6MolecularCell
SciencesResearchCentre,St.George’s,UniversityofLondon,London,UnitedKingdom,7AlanLyellCentreforDermatology,Glasgow,UnitedKingdom,8Departmentof
MolecularandCellBiology,UniversityofConnecticut,Storrs,Connecticut,UnitedStatesofAmerica
Abstract
Thehighmortalityofmelanomaiscausedbyrapidspreadofcancercells,whichoccursunusuallyearlyintumourevolution.
Unlike most solid tumours, thickness rather than cytological markers or differentiation is the best guide to metastatic
potential.Multiplestimulithatdrivemelanomacellmigrationhavebeendescribed,butitisnotclearwhichareresponsible
forinvasion,norifchemotacticgradientsexistinrealtumours.Inachamber-basedassayformelanomadispersal,wefind
that cells migrate efficiently away from one another, even in initially homogeneous medium. This dispersal is driven by
positivechemotaxisratherthanchemorepulsionorcontactinhibition.Theprincipalchemoattractant,unexpectedlyactive
across all tumour stages, is the lipid agonist lysophosphatidic acid (LPA) acting through the LPA receptor LPAR1. LPA
induceschemotaxisofremarkableaccuracy,andisbothnecessaryandsufficientforchemotaxisandinvasionin2-Dand3-D
assays. Growth factors, often described as tumour attractants, cause negligible chemotaxis themselves, but potentiate
chemotaxis to LPA. Cells rapidly break down LPA present at substantial levels in culture medium and normal skin to
generate outward-facing gradients. We measure LPA gradients across the margins of melanomas in vivo, confirming the
physiological importance of our results. We conclude that LPA chemotaxis provides a strong drive for melanoma cells to
invadeoutwards.Cellscreatetheirowngradientsbyactingasasink,breakingdownlocallypresentLPA,andthusforminga
gradientthatislowinthetumourandhighinthesurrounding areas.Thekeystepisnotacquisitionofsensitivitytothe
chemoattractant, but rather the tumour growing to break down enough LPA to form a gradient. Thus the stimulus that
drivescelldispersal is notthe presenceofLPAitself, butthe self-generated,outward-directed gradient.
Citation:Muinonen-MartinAJ,SusantoO,ZhangQ,SmethurstE,FallerWJ,etal.(2014)MelanomaCellsBreakDownLPAtoEstablishLocalGradientsThatDrive
ChemotacticDispersal.PLoSBiol12(10):e1001966.doi:10.1371/journal.pbio.1001966
AcademicEditor:AndreLevchenko,YaleUniversity,UnitedStatesofAmerica
ReceivedFebruary24,2014;AcceptedSeptember5,2014;PublishedOctober14,2014
Copyright:(cid:2)2014Muinonen-Martinetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:ThisresearchwasfundedbyCancerResearchUKcoregrantstoRI,LM,andOS;bytheWellcomeTrust(fellowship095186/Z/10/ZtoAMMand
programmegranttoDB);andbytheBBSRC(coregranttoMJOW).Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,or
preparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
Abbreviations:EGF,epidermalgrowthfactor;FBS,fetalbovineserum;LPA,lysophosphatidicacid;PDGF,platelet-derivedgrowthfactor;RGP,radialgrowth
phase;SCF,stemcellfactor;SEM,standarderrorofthemean;VGP,verticalgrowthphase.
*Email:[email protected]
Introduction size alone. Metastasis is therefore an important, and under-
medicated, potential target forcancer therapy[4,5].
Melanoma Metastasis
Melanoma is an unusually aggressive cancer, which often Melanocyte Migration during Development
metastasizesearlyduringtumourdevelopment[1].Tumoursthat One principal reason behind the aggressiveness of melanoma
have not clinically metastasized are frequently curable, but derives from the developmental history of melanocytes, the
patients arefarlesslikelytosurvive iftumourshavemetastasized pigment producing cells in the skin that mutate to form
beforetheyaresurgicallyremoved,andmetastasisistheprincipal melanomas. During mammalian development melanoblasts, the
cause of cancer mortality [2]. The most influential prognostic melanocyte precursors, emerge from a restricted location at the
factor in predicting metastasis and survival is the thickness of the neural crest, and migrate rapidly from there throughout the
tumour(the‘‘Breslowdepth’’)[3].Thereisadramaticincreasein developing dermis, before maturing into melanocytes on the
the risk of metastasis with only millimeter increases in Breslow basementmembraneoftheepidermis[6].Thusasubstantiallevel
depth [3]. This characteristic is unlike most solid tumours, in of cell migration is required for even skin pigmentation. Even in
which the cytological morphology of the tumour cells and the adults—forexamplefollowingtreatmentforvitiligo—melanocytes
individual genes mutated in the cancer are more important than can spread significant distances from the hair follicles to
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Self-generatedLPAGradientsDriveMelanomaDispersal
[14]).Potentialattractantsformextremelysteepandrathershort-
Author Summary
lived concentration gradients, unlike the physiological conditions
Melanoma is feared because it spreads very rapidly when theassayaimstoreproduce.Moreseriouslystill,conditionseither
tumours are relatively small. It is not known why this side of the filter may be discretely different; cells may grow,
metastasisissoefficientandaggressive.Inparticular, itis survive, or adhere better on one side of the filter than the other,
notknownwhatdrivesmelanomacellstostarttomigrate giving changes in the numbers of cells that can be artifactually
outfromthetumour.Here,wehavestudiedthechemical interpreted as chemotaxis. Direct viewing chambers, such as
signals that guide the migration of melanoma cells. We Dunn,Zigmond,orInsallchambers,aremorelaborioustousebut
find that a component of serum, lysophosphatidic acid
yielda farhigherquality ofdata, with fewerartifacts[15–17]. In
(LPA),functionsasaremarkablystrongattractantforallof
work described here, we use direct-viewing chambers to identify
the melanoma cells that we examined. We also observe
lysophosphatidicacid(LPA)asafarmorepotentchemoattractant
thatmelanomacellsrapidlybreakdownLPA.Weconclude
formelanomacellsthanotherpreviouslydescribedattractants.We
that melanomas create their own gradients of LPA, with
have developed and refined two direct-viewing assays to assess
low LPA in the tumour and high LPA outside. Since
mechanisms of cell dispersal and chemotaxis, allowing us to
melanoma cells are attracted by LPA, this LPA gradient
distinguish chemotactic from chemokinetic and contact-driven
around the melanomas serves as a signal that drives the
tumour cells out into the surrounding skin and blood responses under defined conditions that minimize artifacts.
vessels. Finally, we show that such gradients exist in a Furthermore, the use of direct-viewing chambers makes compar-
mousemodelofmelanoma.Self-generatedLPAgradients isonof attractants’ relative efficienciespractical.
are therefore an intriguing new driver for melanoma
dispersal. The Source of Attractant Gradients In Vivo
The suggested role of chemoattractants in cancer dispersal—
whether growth factors, chemokines, or LPA—raises the crucial
repopulate the surrounding skin. The melanocyte lineage is thus question of how gradients are generated. Chemotaxis will only
inherently migratory. work with signals that are presented as gradients—homogeneous
However, several questions about melanoma progression signals contain no directional information—and the steeper the
remainunanswered.Thefirstiswhatdrivesmelanomastochange gradient, the more efficient the chemotaxis. Chemical gradients
from the relatively benign radial growth phase (RGP) to the far are typically effective over distances of less than a millimetre—
moreinvasiveverticalgrowthphase(VGP)(seeschematicdiagram limits on the efficiency of diffusion make larger gradients
inFigure 1A).In RGP melanomas, cellsonly spread horizontally impractical [18]. Thus for a gradient to be formed there must
along the basement membrane, compared to VGP melanoma bea gradient sourcethat isclosetothetumour.
cells, which are also capable of spreading both upwards into the Alternatively, local gradients may be formed from signals that
epidermis(Pagetoidspread)anddownwards,intoandthroughthe are widely produced, but are absorbed or broken down locally.
dermis(invasion).Thisspreadraisestherelatedquestion,ofwhat This local depletion mechanism is potentially just as effective as
drives cells to migrate away from the primary tumour. Simple, local production, but less often invoked. In the cancer literature,
random migration is an extremely inefficient way of dispersing onlylocalisedsourcesaretypicallyinvoked,forexampleindividual
cellsandalsounlikelytodrivecellstoinvadethroughmatrixand macrophageswithinthevasculatureattracting cancercellswithin
basement membranes. Chemotaxis—cell migration directed by thetumour [10].
gradients of soluble signalling molecules—is implicated as an Ifcellsthatarerespondingtoastimulusarealsoresponsiblefor
importantdriverofmetastasisbyawiderangeofdata[7,8],andis breaking it down, the result is a self-generated gradient. Under
considered necessary to drive efficient invasion. In breast cancer, these conditions the gradient is always oriented away from the
forexample,sometumourcellsmigratetowardsepidermalgrowth current location of the cells. One such example has been shown
factor(EGF)[9].However,EGFgradientshaveonlybeeninferred during the development of the zebrafish lateral line primordium
invivo,nevermeasured,andtheirsourcesareusuallyunclear.In [19–21], in which a dummy receptor locally absorbs an SDF-1
the case of breast cancer, the EGF is thought to be secreted by stimulus to set up a gradient that is detected by a different
macrophagesrecruitedinaparacrineloopbythetumour[10],but receptor. In this work we find that melanoma cells self-generate
for other attractants and cell types the sources of chemotactic chemotactic gradients from unlocalised, exogenous LPA. These
signals are not known. gradients tend to direct cells to disperse outwards from tumours,
In the melanoma literature, most chemotaxis is attributed to thusdirectlypromotingmetastasis.Furthermore,wemeasureLPA
growthfactorssuchasplatelet-derivedgrowthfactor(PDGF)and gradients across real melanomas in vivo. Since melanomas of
EGF [11] and the CXCR4 ligand SDF-1 [12], though a wide sufficientsizebothgeneratetheirownLPAgradientsandrespond
varietyofpotentialattractantshavebeendiscussed[13].Gradients to them, chemotaxis-steered spread of melanomas is almost
of growth factor or SDF-1 have not been identified in vivo, they inevitable.
can only be inferred from the cells’ behaviour or pattern of
responses invitro.
Results
Chemotaxis and Invasion Assays Density-Dependent Outward Migration of Tumour Cell
Chemotaxis assays are typically performed in transwell cham- Toexaminethesignalsthatdrivethespreadofmelanomacells,
bers,inwhichcellsaregrownononesideofamembranefilterand wesetup2-Dassaysfortumourcellspreadusingadirect-viewing
potential attractants are added to the other side. Chemotaxis is chemotaxischamberthatallowsdetailedanalysisofcellmigration
assayedbythenumberofcellsobservedonthefarsideofthefilter [15].Thechambercontainstwowells,connectedbyabridgethat
after a fixed interval. These assays are subject to a wide range of allows diffusion of attractants but not flow. Both cells were
artifacts. Cells’ behaviour during chemotaxis cannot be studied, homogeneously filled with complete medium, but cultured
which makes it extremely difficult todistinguish chemotaxis from melanoma cells [22] were only seeded in one well, at a range of
directionless changes in migratory behaviour (i.e., chemokinesis different densities.
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Figure1.Density-dependentdispersalofmelanomacells.(A)Schematicshowingthestagesofmelanomaspread.(B)WM239Ametastatic
melanomacellsdispersinginuniformmedium.26104cellswereintroducedintoonereservoirofanInsallchambercontainingcompletemedium
with10%FBSthroughout,andobservedbytime-lapsephasecontrastmicroscopy.SeeMovieS1.Theleftsideofeachimageshowsthereservoir
containingcells,whiletherightsideistheviewingbridgeofthechamber.(C–D)Migrationisdensity-dependent.WM1158metastaticmelanomacells
wereseededatdifferentdensitiesinfullmediumwith10%FBS,andobservedasbefore.At26104cells/wellandabove,peakmigrationdistances
increasesharply,asconfirmedbythedistanceat17hours(D;graphshowsmean6SEM).(E)Migrationisnotdrivenbyproductionofarepellent.
26104WM1158cellswereintroducedintoachamberinminimalmediumwithoutserumandobservedat17hoursasbefore.Cellssurviveand
adhere,butdonotdisperse.(F)Migrationisnotdrivenbyproductionofaserum-derivedrepellent.26104WM1158cellswereintroducedintoa
chamberinminimalmediumwithoutserumandobservedat17hoursasbefore.Cellsdisperselessefficientlyinconditionedmediumthaninfresh
medium.(G)Migrationmediatedbychemotaxisupaserumgradientissimilartodensity-inducedmigration.Leftpanel:26104WM1158cellswere
introducedintoachamberinthepresenceofagradientfrom0%FBSaroundthecellsto10%intheoppositereservoir[15].Thecellsrapidlymigrate
towardsthewellcontainingserum.Rightpanel:similarassaywith10%seruminbothreservoirs.PanelstakenfromMoviesS3andS1,respectively.
doi:10.1371/journal.pbio.1001966.g001
Our initial results were surprising: Cells consistently spread create a gradient that consistently directs migration away from
outwards from the well in which they started, even in uniform themselves. We considered that homogeneous attractants would
mediumwithoutanexternallyappliedgradient(Figure 1B;Movie mostlikelyderivefromtheserumaddedtofullmedium.Tofindif
S1). This effect was density-dependent; cells plated at 26103 or dispersalusedarepellentoraconsumedattractant,wecompared
66103cells/wellbarelymigrated,while26104cells/wellmigrated celldispersalinserum-freeandnormalmedium.Cellsinserum-free
up to 350mm in 24hours (Figure 1C and 1D). This behaviour medium are healthy and motile in control movies, but do not
strikinglyresemblesthebehaviourofrealmelanomas,inwhichthe migrateawayfromoneanother(Figure1E),demonstratingthatthe
chanceofmetastasisismorecorrelatedwithtumourthicknessthan cells do not secrete chemorepellents. We also compared cells
any otherparameter [3]. movingoutoffreshmediumintoserum-freeandfullmedium.Cells
This type of density-dependent spreading requires individual dispersed far more efficiently into the rich medium (Figure1F),
cells (or small clusters of cells) to migrate away from the bulk implyingthattheyaredrivenbyattractantsinfreshmediumrather
population. This dispersal occurred in our assays; cells moved thananinhibitorwhoseproductiondependsonserum.
directly away from the well they resided in with unprecedented To test whether consumption of a component of serum
accuracy (Movie S1). This directed, non-random migration can producesapositivechemotaxisresponse,wecomparedmigration
onlyoccurifthemovingcellsperceiveadirectionalcuefromthe in uniform serum to an assay in which cells are exposed to a
bulk populationof thecells to spread. We therefore analyzed the gradientbetweenserum-freemediumandmediumsupplemented
nature of the signal that was directing cells away from the bulk with10%serum(MovieS3).Wefoundthatbothassaysproduced
population.Themostprobablesignallingmechanismsarecontact similar directed migratory responses; cells migrated towards the
inhibition of migration [23] or chemotaxis. We therefore opposite well with or without a preformed serum gradient
examined these potential mechanismsinturn. (Figure 1G). This finding further supports the concept that the
Contact inhibition (of migration, as opposed to the more outward migration is driven by positive chemotaxis, most likely
frequently described contact inhibition of growth) is an effective towards a chemoattractant globally present in the serum but
mechanism for short-range dispersal in which cell:cell contact depleted around thecells.
directs cells away from one another. It has been shown in other We tested this hypothesis using a more traditional chemotaxis
neural crest-derived cell types [24]. However we found no assay,inwhichcellsarespreadhomogeneouslyoverthefieldatthe
evidence to suggest it drives cell dispersal in our assays. Movie start of the assay, giving the cells the opportunity to move in any
S2showsoneexampleinwhichcellsspreadbothindividuallyand direction [14]. We loaded cells into the chamber in complete
whilecontactingoneanother.Somecellssteeraccuratelyoutwards mediumthathadbeenconditionedbymelanomacellsfor48hours,
through multiple cycles of new pseudopods independently of thenreplacedthemediuminonewellwithfreshmediumcontaining
cell:cell contact. Others continue to migrate outwards when 10% serum. The cells migrated towards the well containing fresh
contactingthecellinfront,wherecontactinhibitionpredictsthese medium very efficiently (Figure2A and 2B), showing that an
cells should reverse into the space behind them. Analysis of the attractantinfreshmediumisconsumedbythemelanomacells.
paths of individual cells (Figure S1) shows that cell-cell contact is We confirmed that chemoattractants are present in normal
notsteeringcells;thepathsofcellsthatarecontactingothers,have serumbyexposingmelanomacells—againhomogeneouslyseeded
recently contacted others, and are not in contact are strikingly inthechemotaxischamber—toexogenousgradientsofserum.In
similar. The one apparent example of contact inhibition (Movie homogeneous serum-free medium the cells were healthy, and
S2, cell 2) changed the cell’s direction but did not improve its migrated, but randomly (Figure 2C). When a gradient of serum
outwardaccuracy.Thuswhilethesecellsmayexperiencecontact wasapplied,thecellsmigratedtowardsthehigherconcentrations
inhibition,weconsideredchemotaxisasthemostlikelymechanism with unprecedented precision (Figure2D); their paths are
steering themaway fromthemainpopulation. overwhelmingly oriented up-gradient, in a manner more usually
Cellscouldgeneratechemotacticgradientstodrivedispersalby associated with neutrophils and Dictyostelium [28] than cancer
either of two mechanisms. They could secrete an autocrine cells, which typically chemotax less accurately [29]. The high
chemorepellent and migrate away from it. We have previously chemotacticindexwasmaintainedthroughoutasustainedperiod,
shownthistobeakeydriverofEntamoebapathogenesis[25],in with narrow and accurate confidence interval, and strongly
which chemotaxis away from ethanol generated by the amoebas significantRayleightest[30]fordirectionalmigration(Figure2E).
themselves causescells tomigrate fromthelumen ofthe gutinto Thus serum contains a remarkably potent chemoattractant for
the walls of the gut and eventually the liver of the patient. melanoma cells.
Alternatively, the melanoma cells could locally break down or We therefore conclude that melanoma dispersal across the
consume a chemoattractant that is produced externally, but chamber is driven by positive chemotaxis towards an attractant
spatially homogeneously [26,27], as seen in the zebrafish lateral thatispresentinserum.Theattractantisbrokendownbythecells
lineprimordium[19,21].Ineithercase,densepopulationsofcells themselves into agradient that efficiently disperses cells.
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Figure 2. Dispersal is due to a chemoattractant present in serum. All panels show data from melanoma cells migrating in chemotaxis
chambers as described [15]. (A–B) Cells migrate from conditioned medium towards fresh medium. WM1158 cells were randomly attached to a
coverslipandassembledinachamberin48hourWM1158cellconditionedmedium.Themediuminonechamberwasreplacedwithfreshmedium,
whiletheotherwasleftalone.Tracksofindividualcellsareshownascolouredlines(A).Cellsmovetowardsthefreshmedium,asshownbythespider
plot(B)showingallcelltracks.(C–D)ExampleimagesshowingWM239Ametastaticmelanomacellsafter21hoursinserum-freemedium(C)anda
0%–10%FBSgradient(D).Colouredpathsshowcentroidtracksfromtime0.(E)Quantitativeanalysisofchemotacticresponses.‘‘Spider’’plots(large
panels), rose plots, mean chemotactic index, and Rayleigh test for directionality are shown for cells in serum-free medium and a 0%–10% FBS
gradient(n.100cellsinthreeindependentexperimentsforbothconditions).SpiderplotsshowstrongchemotaxisinFBSgradients;inserum-free
mediumonlyrandommovementisseen.Roseplotsshowoverallmovementfrom6–12hours;theproportionoftotalcellsineachsectorisshownon
a log scale, with red lines representing the 95% confidence interval. The majority of cells in the FBS gradient move in the direction of the
chemoattractant.Rayleightestsstatisticallyconfirmedthishighlysignificantunimodaldirectionality.Graphsofchemotacticindexweregenerated
fromthesamedata.
doi:10.1371/journal.pbio.1001966.g002
Chemotaxis during Tumour Progression physiologically appropriate behaviour (Figure 3A) [22]. Surpris-
Onepotentialexplanationforcancercellsbecomingmetastatic ingly, all the lines we examined responded chemotactically to
is that they evolve chemotactic competence as the tumours serum gradients (Figure3B). Cells from metastases were more
develop [13,31,32], and thus move from unsteered to steered motile than cells from earlier stages (Figure 3C); highly invasive
migration. We therefore examined the ability of a panel of cell (VGP)cellswereslightlymoreaccurate,butnotsignificantlyfaster
lines isolated from different tumour stages and selected for thanthebiologicallyearlier,RGPcells.Cellsfrommoreadvanced
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tumours responded more robustly, but the progression from LPA Is the Dominant Attractant in Serum in 2-D and 3-D
nonmetastatic to metastatic was not marked by the cells newly Assays
acquiring responsiveness—all lines examined were chemotactic
To examine whether LPA was the principal attractive
enough to spread away from the tumour efficiently in the
component of serum, we assayed chemotaxis in the presence of
presenceofanappropriategradient.Severallinesofdatasuggest
theantagonistKi16425,whichspecificallyinhibitsbindingtoLPA
that genetic and epigenetic changes during progression from
receptors 1 and 3 [38]. The effects were again remarkably clear.
RGPtoVGPincreasecells’abilitytosurvive[33];ourdataimply
10mM Ki16425 blocked cell spread in our original, density-
that it is cell survival, rather than chemotactic sensitivity, that
dependent assay (Movie S4) and chemotaxis towards 10% serum
defines the difference. The increase in migratory ability could
(Figure 5A;MovieS5),reducingthechemotacticindexfrommore
modulatecells’abilitytoescapefromaprimarytumour,butour
than +0.4 to zero (Figure 5B). Ki16425-treated cells were
principal conclusion is that melanoma cells from all stages are
obviously healthy, and moved similarly to untreated cells, with
chemotactic.
similar track lengths, showing that the treatment was not making
thecellsnonspecificallysickornon-motile.KnockdownofLPAR1
Identifying the Chemoattractant in Serum
bysiRNAhadasimilareffect(FigureS2A),showingthatLPAR1
There are multiple reports of chemotaxis driving metastasis of is the key receptor for this process, and 10mM Ki16425 also
melanoma and other tumour cells, in particular breast cancer. blocked chemotaxis towards pure LPA (Figure S2B). Again, LPA
Published accounts of chemotactic invasion most often describe chemotaxis is not tumour stage-specific; Ki16425 blocked
growth factors as the attractants—for example EGF for solid chemotaxis in all cell lines from all stages of cancer progression
tumours [34], and EGF, hepatocyte growth factor (HGF), and
(Figure 5C). RGP and VGP cell lines were completely inhibited,
stem cell factor (SCF)/KitL for melanoma [13]. However these
andthehighlymotilemetastaticlinesweresubstantiallyinhibited.
attractants were often identified in transwell chambers, which as
The residual chemotaxis in the presence of inhibitor could
earlier discussed are subject to a range of artifacts, in particular
represent either incomplete inhibition by the antagonist, or a
false positive. For example, the positive well might promote
smallamountofchemotaxistoanotheragent.Fromthesedata,we
survival, growth, or adhesion of cells that move randomly across
concludethatLPAisoverwhelminglythedominantchemoattrac-
the membrane. Our direct-viewing chambers provide a far more
tant inserum forall linesexamined.
rigorousanalysis.Wethereforetestedabroadrangeofattractants
While chamber-based assays are optimized to allow accurate
in our assays. To our surprise, no growth factor acted as an
anddetailedrecording,theyprovidea2-Dviewofaprocessthat
attractanttoanymeasurabledegree(Figure 4A);steeporshallow
more often happens in 3-D tissues in vivo [39]. We therefore
gradients gave no obvious movement upgradient, and no
examined the role of LPA in a widely used organotypic tumour
significant chemotactic index towards any growth factor tested
cellinvasionmodel[40].Inthissystemmelanomacellsareadded
(Figure 4B). We therefore conclude that the chemotaxis towards
to the top of a plug of collagen in which fibroblasts are growing,
serumweobservedwasunlikelytobetowardsgrowthfactors.This
and over time they migrate vertically downwards into the 3-D
does not, of course, demonstrate that melanoma cells are never
matrix.Duringthecourseoftheassay,thecollagenplugissetso
chemotactic towards growth factors; but it clearly shows the
only its bottom face contacts the medium, at which point
surprisingandefficientchemotaxistowardsserumobservedearlier
malignant melanoma cells invade downwards [41]. We hypoth-
ismediated byanother molecule.
esized that the melanoma cells were driven by a self-generated
EGFandPDGFdidincreasecells’speed(Figure 4C),butthey
LPA gradient as in Figure 1B, once fresh LPA could only be
did not provide directional specificity. They therefore acted as
supplied fromthebottom. Thishypothesis issupported by assays
chemokines, regulating overall cell behaviour, rather than as
inwhichthecollagenplugsremainsubmerged,andnoinvasionis
chemoattractants that could steer thecells.
seen (Figure S3), further rejecting contact inhibition of migration
The striking accuracy of chemotaxis demonstrated by melano-
as a mechanism of invasion. When the gels were treated with
ma cells towards serum was more reminiscent of neutrophil
Ki16425, themelanomacells did not invadedownwards intothe
chemotaxis towards formyl peptides, or Dictyostelium towards
gel (despite comparable numbers of cells at the end, showing no
cAMP, which signal through G-protein coupled receptors
changeingrowthorsurvival).Quantitativeanalysisconfirmsthat
(GPCRs) rather than growth factor receptors like EGFR and
Ki16425 strongly inhibited invasion in both cell lines that were
PDGFR. We therefore investigated SDF-1, the ligand for the
invasiveinthisassay(Figure 5Dand5E).ThusLPAisadominant
GPCR CXCR4, which has been associated with poor prognosis
steering system for 3-D organotypic assays, as well as for 2-D
and malignancy of melanoma [35]; but again, it was not
chamber assays.
measurably attractive to cells in our assays (Figure 4B, compare
with strongresponse toserum).
Melanoma Cells Break down LPA to Form Outward-
However, LPA, another well-known component of serum that
signals through GPCRs, was strikingly attractive to melanoma Facing Gradients
cells. A gradient from 0 to 1 mM LPA across the chamber Ourearlierdata(Figures1Band2A,inparticular)showedthat
(consistent with the approximate concentration of LPA in serum; melanoma cells disperse by depleting a chemoattractant from
see below) induced chemotaxis almost as effectively as 0%–10% serum. We therefore tested whether melanoma cells are able to
serum (Figure 4D), yielding a comparable chemotactic index deplete LPA from their surroundings. Full medium with and
(Figure 4E). This was a surprise: LPA is more typically described withoutserumwasincubatedwithdifferentdensitiesofmelanoma
as an inflammatory mitogen, acting on haematopoietic cells such cells for different times, then LPA was extracted from the
asmacrophages.Itappearsfrequentlyinthecancerliterature,but conditioned medium and analyzed by mass spectrometry [42].
moreoftenasamitogenandchemokineforcancercells,actingvia This confirms that the melanoma cells effectively break down
autotaxin, whichcatalyzes theproduction of LPA fromlysophos- LPA; the conditioned medium was depleted in a density-
phatidylcholine [36]. However in our assays the chemotaxis of dependent manner (Figure 6A) and in a timescale that correlates
melanomatoLPAwasagainremarkablyaccuratecomparedwith withthemediumconditioningexperiments inFigure 2Aand2B.
theweaker chemotaxistypically seen incancer cells [37]. Oneadvantageofusingmassspectrometryistheidentificationof
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Self-generatedLPAGradientsDriveMelanomaDispersal
Figure3.Chemotaxisofcellsfromdifferentmelanomastages.(A)Chemotaxisofapanelofsixcelllinesfromdifferentmelanomastages
(RGP,green;VGP,purple;metastatic,red)upa0%–10%FBSgradientwasmeasuredasabove(n$45cellspercellline).(B)Chemotacticindexofcells
fromdifferentstages.Datafrom(A)werecollatedbymelanomastage.Chemotaxisimprovesasthestageofmelanomaprogresses,althougheven
theearliestRGPcellsshowclearchemotaxis.(C)Speedsofcellsfromdifferentstages.Datafrom(A)werecollatedbymelanomastage.Metastatic
lines are conspicuously faster (p-values from unpaired t-tests), although again the speed of RGP and VGP cells is still relatively high for non-
haematopoieticcells.
doi:10.1371/journal.pbio.1001966.g003
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Self-generatedLPAGradientsDriveMelanomaDispersal
PLOSBiology | www.plosbiology.org 8 October2014 | Volume 12 | Issue 10 | e1001966
Self-generatedLPAGradientsDriveMelanomaDispersal
Figure4. Identification of LPA,ratherthan growthfactors, as the principalattractantin serum.(A) WM239Acells wereexposed to
gradientsoflow(light)andhigh(dark)concentrationsofseveralgrowthfactorsandthechemokineSDF-1incombinationwithSFM.SpiderandRose
plotswithRayleightestsareshown(n.40cellsforeachcondition).ConcentrationstestedwereEGF(6.25and25ng/ml),PDGF(25and100ng/ml),
HGF(10and30ng/ml),SCF(10and100ng/ml),andSDF-1(100and300ng/ml).Noneshowsobviouschemotaxis.(B)Quantificationofdatafrom(A).
Serumgradientspromotestrongchemotaxis(p,0.0001,unpairedt-test),butgradientsofallgrowthfactorstestedshownosignificantchemotactic
index (p$0.40). (C) Growth factors enhance cell speed. Data quantitated from the cells in Figure3A. Directionless cell speed was measured by
totallingthedistancemovedbetweentimepoints.EGFandPDGFstimulatecellsinserum-freeminimalmediumtospeedscomparablewithserum
gradients. Single asterisk: Different from SFM alone, p,0.001, unpaired t-test; double asterisk: p,0.0001). (D) LPA and serum drive comparably
efficientchemotaxis.WM239Acellswereexaminedinachamberrespondingto0%–10%FBSand0–1mMLPA.Thespiderplotshowssimilarcellular
responses to the two gradients. (E) Quantitative analysis of chemotaxis towards LPA and serum. Chemotactic index was calculated from three
experimentsincludingthatshownin(D).Cellsrespondcomparablytobothconditions.BarsshowSEM.
doi:10.1371/journal.pbio.1001966.g004
molecular subspecies. The biological activity of LPA is known to still in the tissues immediately outside the tumour (Figure 8D).
vary with its structure [43,44]. In particular, there is a strong Cells at the edges of the tumour are therefore experiencing an
correlation between biological activity and the degree of poly- outward-oriented LPA gradient tending to drive them out into
unsaturation, and also acyl chain length [45]. Melanoma cells surrounding tissues andvasculature.
broke down the biologically active species more rapidly than the WefurtherexaminedtheLPAspeciesinthetissue.Formsthat
others (Figure 6B), ensuring that the most active species also arestronglyassociatedwithsignalling,inparticular18:2-LPAand
formed thesteepest gradients. 20:4-LPA [48], formed the steepest gradients (Figure 8E), while
gradients of non-signalling forms such as 18:0-LPA were flatter.
The Role of Growth Factors This finding further supports the idea that the gradients of LPA
The results we have obtained conflict with the established are specifically produced assignals targeted atLPA receptors.
dogma that growth factors are primary melanoma chemoattrac- This study is, to our knowledge, the first time a chemotactic
tants [13]. To reconcile these accounts with our data, we gradient has been directly measured around tumours in vivo.
examined the role of growth factors during chemotaxis towards Thereareanumberofsituationswherethepresenceofagradient
LPA. As shown previously (Figure4C), EGF and (particularly) hasbeeninferredfromcellularbehaviour,mostprominentlyinthe
PDGF increased the basal speed of cells. Gradients of EGF and paracrine loops shown by Segall and others [10]. However, such
PDGF, and mixtures of both, enhanced the accuracy of gradientsmustbydefinitionbelocalandtendtobetransient.The
chemotaxis to LPA (Figure 7); LPA, EGF, and PDGF together gradients we observe in melanomas are clear, large-scale, and
in serum-free minimal medium were as effective as 10% serum. provide a convincing driver for cell dispersal, and one highly
Mosttellingly,however,whencellswerepresentedwithLPAand plausibleexplanationofwhymelanomasaboveacertainsize,and
growth factor gradients oriented in opposite directions, they hence Breslowthickness, always tendtobemetastatic.
chemotaxed towards the LPA not the growth factors; if anything
they migrated towards the LPA with enhanced efficiency Discussion
(Figure 7B, bottom two lines). Thus when examined in the high
levels of detail afforded by our chambers, the growth factors are In this work, we have shown that LPA is a potent chemoat-
potentiallyimportantaccessoryfactorsthatincreasecellspeedand tractantformelanomacellsingeneral,andthatoutward-oriented
efficiency of chemotaxis, but they do not themselves act as gradients of LPA are self-generated by melanoma cells. Because
chemoattractants. self-generated gradients are always oriented away from tumours,
Theseresultsarereminiscentofobservationsofdevelopmentin this combination provides a plausible mechanism for driving
vivo,inwhichthegrowthfactorSCFpromotesmigrationbutnot tumourcelldispersal.Wedonotexcludeothermechanisms;ithas
direction of melanoblast migration [46]. It is possible that the for example been proposed that LPA regulates cadherin levels
melanoma chemotaxis to growth factors observed in other work [49], which would not be visible in our assays. Growth factor
[13] is due to changes in speed alone, which as discussed earlier chemotaxis may be visible under the appropriate conditions
can cause a false positive in transwell assays. It has also been (though, as discussed previously, many data are from transwell
shown that growth factors can cause cancer cells to secrete LPA assays, which are artifact-prone and unreliable). Likewise, we do
[47],whichcould alsoprovideanelement ofindirectchemotaxis notexcludeothermechanismsthanchemotaxis.Contactinhibition
in manytypesof assay. ofmigrationoccursinmanycelltypesderivedfromtheneuralcrest
andsoisprobablyfoundinmelanoma,anddefectsincellgrowth
LPA Gradients in Tumours In Vivo and survival in inappropriate locations are of course important
factors. But the mechanism we have found that overwhelmingly
WehaveclearlyshownthatLPAisapotentchemoattractantfor
dominates the dispersal in our assays is robust and is apparently
melanomacellsofallbiologicalstages.Todeterminewhetherthis
chemotaxis was an important driver of melanoma chemotaxis in activeinahighproportionofmelanomas.Itisthereforelikelytobe
vivo, we investigated whether the tissue surrounding real a particularly important mediator of tumour cell dispersal. We
melanomas contained LPA gradients that would direct cells out hypothesize that similar mechanisms will be common in cancer
of tumours. Mice that are heterozygotes for the driver mutation metastasis.
BrafV600E (the most prevalent driver of human melanomas) and
deletion of the tumour suppressor PTEN develop sporadic The Source of LPA
melanomas (Figure 8A) genetically and cytologically comparable The source of LPA around melanomas is unknown. In many
tohumantumours(Figure 8B).Wetookpunchbiopsiesfromthe tumours, including melanoma, expression of autotaxin and thus
tissue in and across melanomas (Figure8C) from several mice, autocrine production of LPA has been associated with tumour
extracted total lipids, and examined LPA levels using mass progression[50].ThisLPAproductionappearstobeamechanism
spectrometry.Inallnon-ulceratedmelanomasweexamined,LPA forpromoting melanomagrowth,rather than drivingchemotaxis
levelswerelowinsidethetumour,higherattheedges,andhigher andinvasion.LPAgeneratedbythetumouritselfwouldbefound
PLOSBiology | www.plosbiology.org 9 October2014 | Volume 12 | Issue 10 | e1001966