tumor initiationg cells

7/30/2019 Tumor Initiationg Cells

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Mdn antian dg disvy gan in th mid-tntith nty ith th svatin that yttxihmthapti agnts d sd t tagt ansith high pifativ ats1,2. Sin thn, th disvyand dvpmnt f an thapis, initiay n thasis f mpiia svatins, has m inasingydpndnt n ndstanding f hman tmigy. Hv, dspit advans that hav d t thdvpmnt f n thapis, tatmnt ptins a stiimitd f many typs f hman an patiayths ith ndiffntiatd phntyps, sh as asastyp ast an and pgnsis mains p.In additin, th aiity t manag tm nand mtastasis fing sssf initia indtin f

missin ntins t a hang.Th xpimnta dmnstatin f tm-initiating

s (ppay knn as an stm s) in svahman tms in nt yas310 sppts tmhiahy as a fndamnta npt in tm igyand pmiss a n a tagt f antian dgdisvy. Athgh th an stm hypthsis asfist ppsd dads ag (vid in Refs 11,12),many aspts f this hypthsis main spativ anda sti vving. A minima patina dfinitin ftm-initiating s is: ths tm s that havth aiity t -g th tm fm hih thy isatd idntifid (fIG. 1), hih impis that th

tm-initiating s an ny dfind xpimn-tayin vivo12. M gnay, tm-initiating s a

vid as ths s at th apx f th tm hiahy(BOX 1), hih highights th f aant diffn-tiatin in tmignsis. Mtiptny f inag diff-ntiatin is iky t a fqnt, t nt a nssay,ppty f tm-initiating s.

Th tm an stm ds nt impy that th is divd fm a nma stm 13. Dpnding ntm typ, th s iginating th tm an stms, pgnit s diffntiatd s, and nd ntmath th phntyp f th vnta tm-initiating ith spt t l-rnwal and diffntiatin apaity(s th fig in BOX 1). Nvthss, th igin f th

tm-initiating s d hav impiatins f ththapti ind f th statgy that is sd t tagtthm. F xamp, kiing nma stm s ang ithth tm-initiating s d ad t a hni ssf nma gnatin, has dstying th nmapgnit s may ss f a ng-tm pm.Anth imptant pint is that tm-initiating sa nt nssaiy a14,15. Mv, th havi andfqny f tm-initiating s d as inf-nd y vais nvinmnta fats. Th fndamntanpt ndying th an stm hypthsis is ntatd t th igin, ast fqny patiaativity v (f xamp, pifatin at) f ths

*Oncology Discovery,

Wyeth Research, 401 NorthMiddletown Road, Pearl

River, New York 10965, USA.Cancer Research, Abbott

Laboratories, 100 Abbott

Park Road, Abbott Park,

Illinois 60064, USA.The Hospital for

Sick Children, 555 University

Avenue, Toronto, Ontario,

M4G 1X8, Canada.

Correspondence to B.-B. S. Z.

and J. C. G.

e-mails:[email protected];

[email protected]

doi:10.1038/nrd2137

Self-renewalTh ability o a cll to

rproduc itl without loing

dvlopmntal potntial,

charactrizd by cll diviion

in which dirntiation

i blockd in at lat on

daughtr cll.

Tumour-initiating cells: challengesand opportunities for anticancerdrug discoveryBin-Bing S. Zhou*, Haiying Zhang, Marc Damelin*, Kenneth G. Geles*,

Justin C. Grindley* and Peter B. Dirks

Abstract | The hypothesis that cancer is driven by tumour-initiating cells (popularly

known as cancer stem cells) has recently attracted a great deal of attention, owing tothe promise of a novel cellular target for the treatment of haematopoietic and solid

malignancies. Furthermore, it seems that tumour-initiating cells might be resistant to

many conventional cancer therapies, which might explain the limitations of these agents

in curing human malignancies. Although much work is still needed to identify and

characterize tumour-initiating cells, efforts are now being directed towards identifying

therapeutic strategies that could target these cells. This Review considers recent

advances in the cancer stem cell field, focusing on the challenges and opportunities

for anticancer drug discovery.

R E V I E W S

806 | ocTober 2009 | VoluMe 8 www.at.m/w/dd

2009 Macmillan Publishers Limited. All rights reserved
mailto:[email protected]:[email protected]


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Differentiate ?

?

?

TICs

Targetniche

Combinationtherapy

DestroyTICs

Selfrenewal

Tumour-initiating cell (TIC)

Niche

Tumour progenitor anddifferentiated tumour cell

Anoikis

A orm o programmd cll

dath that i inducd in

anchorag-dpndnt cll

whn thy bcom dtachdrom th urrounding

xtracllular matrix.

Niche

Cll and/or xtracllular

matrix componnt in pciic

anatomical location that

rgulat th participation o

th normal tm cll in tiu

gnration, maintnanc and

rpair. In om ca, th

bhaviour o tumour-initiating

cll might alo b inluncd

by intraction with

urrounding cll and matrix.

s16. Stdying an ppatins ith npts fstm igy in mind is iky t ing fth insightint ma dg tagts and inia statgis.

cnt fai ith an tatmnt is nt sayd t a ak f pimay spns initia indtinf missin, t t aps tm n aftthapy, in hih tm-initiating s a thght thav ia s. A maj hang n is t disvagnts and statgis that tagt an and tmaps at thi appant s. Fing an vvi

f th nt stats f th an stm hypthsis,this ati thf fss n th hangs andpptnitis f antian dg disvy.

Cancer stem cells: evidence and controversy

In th mid 1990s,Jhn Dik and ags dmn-statd th xistn f tm-initiating s in atmyid kamia (AMl) sing th nn-s diatisv mind immndfiint (NoDScID)ms md3,17,18(BOX 2). Th pifid ppatins fkamia-initiating s ntaind a hmsmatansatin that as idntia t that fnd in thipgny, th ast s. As as sppting th

ngni nat f kamia, this imptant findingpintd t th ganizatin f kamia as a stm hiahy19.

Simia stdis a mh m diffit f sidtms. Sid tms ntain th tm s and

vais stma s, and aking th intatinstn s in sh a tm might ind anoiki hang th pptis f th tm s. Fthm,assays that invv injtin f s int a n tiss a-tin in mi may fai t apitat th nvinmnt fths tm s in th igina tms. Nvthss,tm-initiating s fm sid tms, indinghman ast, ain, n, panati, iv and va-ian an and manma, hav n sssfy is-atd sing apppiat sfa maks, indingcD44, cD24, cD133, pithia adhsin m(ePcAM) and ATP-inding asstt s-famiy bmm- 5 (Abcb5)410. Ths tm-initiating s an p-d phnpis f th igina pimay tms hntanspantd int NoDScID mi.

Thtia and thnia qstins main gading

hth th s isatd a th t and ny tm-initiating s that fntin in th sid tms fpatints20. cnt sting pts a thght tfav nich-indpndnt tm-initiating s (BOX 2),hih may sm s in ativatd miizd statsthat a std hn tanspantatin assays a p-fmd n nma stm s21. ctain maks sd fpsptiv sting may as st f s that vadth immn systm22, hih d as a vantppty f tm-initiating s. Nvthss, anm f maks sd in sting a mging asing pditiv f disas pgssin9,23, indiating thatthy idntify iniay imptant ppatins.

It as ntid that sm xpimnta ms mdsf kamia d nt f th an stm hypth-sis, sggsting that tain hman ans may ntadh t this md14. Hv, as ith many insthat hav st th hiahia stt f th pimaykamia fm hih thy iginatd, sm xpi-mnta ms mds may nt aaty ft thtm htgnity and pathgia nvinmnt fspntansy ing hman maignanis16. It asas agd that tain phntypiay distint ppa-tins f hman ans fai t g hn injtd intimmndfiint mi as f diffns in thms and hman minvinmnt 20, s th idn-tifiatin f tm-initiating s in ms mds

adds diiity t th an stm hypthsis2432.Fthm, tansgni mds faiitat inag-taing xpimnts, hih an pvid vidn ftm initiatin and tm hiahis itht thimitatins and xpimnta vaiaiity f tanspanta-tin assays30,31.

Athgh ging vidn fm a id ang fsystms favs th xistn f tm-initiating s,th may nvthss divsity f tm hiahyin diffnt patints and vaying dgs t hih diff-nt tm-initiating s a stm -ik. Many an-s might ntain sppatins f tm-initiatings, t sm d ntain mmn tmigni s,

Figure 1 | T a tm t ad tat tat t tat

tm-tat . According to the cancer stem cell hypothesis, tumour cells

are heterogeneous and only the tumour-initiating cells have the ability to proliferate

extensively, give rise to differentiated cells and form new tumours. There are various

therapeutic strategies that could target tumour-initiating cells. Killing these cells could

be achieved by inhibiting their survival pathways (for example, with inhibitors of

phosphoinositol 3-kinase or interleukin-4-specific monoclonal antibodies), or sensitizing

them to chemotherapeutic agents (for example, with a checkpoint kinase inhibitor).

Some of the survival pathways that are used by tumour-initiating cells could also be used

by the bulk of the tumour, so agents targeting these pathways are expected to kill more

than just tumour-initiating cells. Alternatively, differentiating the tumour-initiating

cells (for example, with bone morphogenetic proteins or CD44-specific monoclonal

antibodies) might be a successful therapeutic strategy, as the bulk of the tumour has

limited proliferation potential. Inhibition of developmental signalling that is involved

in self-renewal (for example, with inhibitors of Wnt, Hedgehog or Notch pathway

signalling) might work by both mechanisms. In addition, anti-angiogenic therapy

might work in part by affecting the vascular niche of tumour-initiating cells. However,

tumour-initiating cells are probably genetically unstable, and a committed progenitor

could regain renewal activity. It might be therapeutically advantageous to combine

agents that target tumour-initiating cells with conventional agents that reduce the

bulk of the tumour and agents that target the niche.

R E V I E W S

NATure reVIewS |Drug Discovery VoluMe 8 | ocTober 2009 |807

http://www.uniprot.org/uniprot/P16070http://www.uniprot.org/uniprot/P25063http://www.uniprot.org/uniprot/O43490http://www.uniprot.org/uniprot/P16422http://www.uniprot.org/uniprot/Q2M3G0http://www.uniprot.org/uniprot/Q2M3G0http://www.uniprot.org/uniprot/P16422http://www.uniprot.org/uniprot/O43490http://www.uniprot.org/uniprot/P25063http://www.uniprot.org/uniprot/P16070


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Normal tissue

Differentiated cells

Stem cell

Multipotent

progenitor

Progenitor

Initial tumour

Tumour progenitors anddifferentiated tumour cells

Tumour-initiating cell

Advanced tumour

Target bulk of the tumour cells

Target tumour-initiating cells

Tissue or

tumourhierarchy

Clonal evolution

Mutations

Mutations?EMT?

Mutations

ith itt hiahia ganizatin15. cntvsy asid,th an stm hypthsis pvids a nv fam-k t stdy a htgnity, aant diffn-tiatin and tmhst intatins in many ans.

Framework for understanding cancer properties

Th an stm hypthsis ds nt ntadit thstaishd na vtin vi f an, t instadsggsts a ky f tm hiahy in tmvtin and highights th imptan f an aantdiffntiatin pgamm in tmignsis (BOX 1). onppa xpanatin f tm htgnity is gntiinstaiity, hih is a mmn fat f ans ad-ing t mphnsiv an gnmi anaysis3335.

cnvsy, a ag pat f th appant mphgiaand phntypi htgnity an xpaind y a-ant diffntiatin, and pignti hangs in tmsd as dynami and nsta36,37 (s th fig inBOX 1). Th havi f tm-initiating s d fth mdatd y tmhst intatins. In thisstin, nsid sm f th ky haatistis ftm-initiating s.

Tumour-initiating cells diverge from their cells of originwith increased self-renewal capacity.Athgh thigins f tm-initiating s may vay, thy shasva pptis ith nma stm s, spiay th

xtnsiv apaiity f sf-na12. If th an stm hypthsis is t, th vtin f th tm isagy th histy f hangs in sf-ning tm-initiating s (BOX 1), and sf-na pathays might m nsvd than sfa maks amng tm-initiating s.

Athgh pathays that gat sf-na a tightyntd in nma stm s, in tm-initiatings thy may nstittivy ativatd imppygatd thgh gnti and/ pignti hangs,ading t nntd gth38. Sva stdis shthat bMI1 pym ing fing ngn (BMI1) andth wnt signaing m -atnin gat th sf-na f hamatpiti stm s (HScs) and th

pifatin f kamia-initiating s3941. Atay,many kamia-initiating s hav a high sf-na apaity than nma HScs3. In additin, it asnty shn that maintnan f tans tm-initiating s is dpndnt n wnt and -atninsignaing 42. Hdghg signaing gats tm-initiating s fm mtip myma43 and hnimyid kamia (cMl)44. Dvpmnta signaingpathays that gat nma stm sf-na,inding wnt, Hdghg and Nth (fIG. 2), hav nshn t ativ in nms hman ans38,45 andmay ady imptant f sf-na in many an-s. Thy as hav imptant s in pgnits and a

Box 1 | Tumour hierarchy and clonal evolution

For decades, tumour initiation and development has been regarded as a multistep process that is reflected by

the progressive genetic alterations that drive the transformation of normal human cells into highly malignant derivatives202.

As cancers arise only after multiple mutagenic events, long-lived cells are probably the most capable of supporting such

cumulative changes. Based on genetic variations already observed in the tumour-initiating cell populations from different

tumours76,203 and genetic mutations in developmental pathways in different cancers34,38, it has been proposed that

progressive genetic alterations might occur at the level of tumour-initiating cells. The clonal progression to cancer could

operate through the stem cell compartment (see the figure), as already shown in leukaemia-initiating cells204.In normal tissues, the heterogeneity of cells reflects a hierarchical programme of differentiation in which multiple mature

cell types are derived from a common multipotent stem cell through intermediate progenitors. Heterogeneous populations

of cancer cells at various differentiation stages could be the result of both acquired mutations and aberrant but hierarchical

differentiation programmes (see the figure). Cancer is both a proliferation and a differentiation disease, and the clonal

evolution and cancer stem cell models might not be mutually exclusive, as initially thought. Owing to genetic instability,

the tumour-initiating cells isolated from a clinically detectable tumour would probably have a substantially different

genetic profile from the initial transformed cells that originated the tumour (see the figure). Also, as chemotherapeutic

agents are often mutagenic, the phenotype and frequency of the tumour-initiating cells in relapsed tumours are expected

to be distinct from those of early-stage lesions. In practice, combination treatment involving both traditional therapies and

therapies that target tumour-initiating cells will probably be required to ablate all cancer cells, particularly as a genetically

unstable tumour cell will present a moving target. EMT, epithelialmesenchymal transition.

R E V I E W S


http://www.ncbi.nlm.nih.gov/gene/648?ordinalpos=3&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSumhttp://www.uniprot.org/uniprot/P35222http://www.uniprot.org/uniprot/P35222http://www.ncbi.nlm.nih.gov/gene/648?ordinalpos=3&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum


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Asymmetrical division

A orm o cllular rplication

in which a cll rnw itl

and gnrat a mor

dirntiatd progny.

Symmetrical division

A orm o cllular rplication

in which a ingl cll giv ri

to two idntical cll.

Epithelialmesenchymaltransition

A cllular program in normal

dvlopmnt and in cancr

whrby cll o an pithlial

origin acquir th proprti o

mnchymal cll, typically

charactrizd by lo o cll

adhion, rprion o

e-cadhrin xprion,

and incrad cll mot ility.

Oncomir

MicroRNA known to

b involvd in cancr

and tumorigni.

id impat n th inag, and d hav additinas in th gatin f tm and stma s. In thisapidy xpanding fid f tm igy, n ts yhih s may aqi nhan sf-na aiitya gay ing disvd, and sf-na s fassia an gns a as mging. F xamp, ygaining tain pignti pgamms sing tainminatins f tm sppss gns, pgnits d gain th apaity f sf-na and mmaignant46,47.

Sf-na an afftd y hangs in pif-atin, diffntiatin and/ apptsis. Aantyinasd sf-na might thf asd y dif-fnt mhanisms, sh as an inasing pifatin at a shift in th aan f divisin fm aymmtricaldiviion t ymmtrical diviion48. Tm-initiating s

fm AMl and cMl a msty qisnt49,50, hastm-initiating s fm many sid tms d pifativ51. Th tmigni ast and pan-ati tm-initiating s hav y pfisthat a simia t ths f k tm s, hihd nt hav tm-initiating aiity, shing it is nta pifativ advantag f ths tm-initiatings v th nn-tmigni s that ads t dif-fntia ngaftmnt4,8. by ntast, sy pifat-ing intstina stm s fm phsphatas and tnsinhmg (Pten)-dfiint mi initiat intstinapypsis52. Th mst imptant and haatistifat f tm-initiating s thf sms t

thi inasd sf-na ptntia, ath than thipifatin at. Indd, ding th na vtinf tm-initiating s, th may stng s-tin f inasd sf-na apaity than f highpifatin53. In additin, a maignant tmigni d hav ndiffntiatd pathgia fatsassiatd ith inasd sf-na, inding mta-stati and p-angigni apaiitis.

Tumour-initiating cells in metastasis and tumourhost

interactions.A ay f th an stm hypth-sis is that maspi mtastass may ais fmmigating dissminatd tm-initiating s54. Inpatints ith ast an, tmigni cD44+cD24/s a adiy dtta in mtastati pa ff-sins, and mst ay-dissminatd an s that

a dttd in th n ma hav a ptativ asttm-initiating phntyp4,55. Intstingy,cD133+cXcr4+ tm-initiating s that a fndat th invasiv fnt f panati tms hav nshn t dtmin th mtastati phntyp f thindivida tm56. Anth hypthsis is that mta-stati tms iginat hn s in a pimay pithiamaignany ndg an pithlialmnchymal tranition(eMT)57. lik sf-na, eMT is gatd y dvp-mnta signaing pathays, sh as th wnt and Nthpathays58,59. expssin f th oncomirLET7in a asttm-initiating md inhiits sf-na, eMTand xngaft mtastasis60. M nty, it as ntid

Box 2 | Mouse models for cancer research and drug discovery

In the mid 1980s, the availability of athymic (nunu) mice and the subsequent development of immunodeficient mouse

strains with other genetic lesions (including severe combined immunodeficient (SCID) mice, which lack both B and

T cells) allowed the widespread use of human tumour xenografts as mouse models for cancer research and drug

discovery205. Xenografts can be established either by injecting human tumour cell lines or by direct implantation

of patient biopsies into immunodeficient mice. The wide range of tumour cell lines and possibility ofex vivo genetic

or pharmacological manipulation before xenotransplantation made human xenograft tumour models popular tools

for discovering and developing not only cytotoxic agents but also tumour-targeted agents.A major drawback of these mouse models is that they have limited utility in measuring tumour self-renewal in vivo,

particularly for tumour cells from primary sources. Models that permit efficient engraftment are a prerequisite for

assays that directly measure tumour self-renewal by serial transplantation. Animal models with an increased efficiency of

engraftment have since been developed. The SCID mouse was crossed with the non-obese diabetic (NOD) mouse, and

the NODSCID progeny can be engrafted by various tumour types and sustain the serial transplantation that is required

for assessing long-term self-renewal potential35. However, NODSCID assays still have limited efficiency of engraftment

and so can lead to underestimations of the frequency of human tumour-initiating cells. Notably, the use of more highly

immunocompromised NODSCID interleukin-2 receptor -chain-null (Il2rg/) mice can increase the detection oftumorigenic melanoma cells by several orders of magnitude15. Although engraftment is improved by implantation into

NODSCID Il2rg/ mice, it is also clear that immune cells have a role in the progression of many tumours. To properly

model certain tumours, disabling the mouse immune system might not be desirable. It may ultimately be necessary to

provide a humanized immune repertoire in mice, perhaps by genetic engineering or haematopoietic cell transplantation.

The limitations of xenotransplantation assays are particularly apparent in analysing tumour-initiating cells from

solid tumours. The current protocol, even with recent improvements 15, is thought to favour niche-independent

tumour-initiating cells, which may not be representative of the population of tumour cells that can exhibit stem cell-likeproperties in their human tumour environment. Orthotopic models might more faithfully maintain the tumourhost

interaction, giving tumour-initiating cells a greater chance to interact with and obtain stimulation from a mouse

environment. Co-injection with tumour-associated stromal cells might be another approach to improve the clinical

relevance of xenotransplantation, and can resolve some of the issues related to the presence of murine stroma in animal

models (fIG. 3b). Avoiding the transplant altogether by generating genetic mouse models of tumours may better

maintain the tumourhost and tumour-initiating cellniche interactions30,31. However, owing to the practical difficulty of

modelling the multitude of genetic changes in a tumour, this strategy may be more suited to studying tumour-initiating

cells that have few defined genetic lesions, perhaps resembling tumour-initiating cells from early-stage human disease.

R E V I E W S


http://www.ncbi.nlm.nih.gov/gene/19211?ordinalpos=5&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSumhttp://www.ncbi.nlm.nih.gov/gene/19211?ordinalpos=5&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum


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that s ndging eMT and tm-initiating ssha many maks and pptis61. Ths findingsva th ptntia pastiity f ths tm s andsggst that th d sm mmn gatypgamms ndying eMT and sf-na (s thfig in BOX 1).

Th havi f nma stm s is tightygatd y signas that th s iv fm thiminvinmnta nihs, hih a pvidd yth adjant s and/ xtaa matix mp-nnts6264. A nih, hi sppting th sf-naand maintaining th idntity f a stm , as n-ts stm nm and pifatin. This ntf nm and pifatin might a pvntativmhanism against an65. Simiay, it is nivathat th tm minvinmnt d as nstaintm-initiating s, as sm tms i dmant dvp sy v dads66. on thy f tmdmany is that tm-initiating s a hd inhk y a nihstm intatin. can dpgss if th nih xpandd atd thgh

gnti pignti mans65. It has n shn that anatatin in a HSc nih an ad t mypifativdisas67. Tm-initiating s d as pfn-tiay aiz t nvinmnts that fav pifatin,sh as vasa nihs6870, vn pmt th fma-tin f thi n nih71. Atnativy, mtatins mightnd tm-initiating s indpndnt f nihsignas, thy ifting nvinmnta nts n sf-na72 and inasing th isk f mtastasis.

Why do many therapies fail to eradicate cancers?

Many patints ith an, patiay ths ith sidtms, ith d nt spnd t xisting an th-apis (inding hmthaptis, adithapy andtm-tagtd agnts) aps qiky aft initiamissin. Ky pssi asns f this fai indth inhnt dg sistan f tm-initiating s,th inffiiny f th tatmnt and/ th gntiinstaiity f an s.

It has n sggstd that th m aggssiv andfaty ans ntain m tm-initiatings73,74. cimstantia vidn in sppt f this n-ntin aady xists in mdia pati. Fingan thapy, a patints tm is xamind tassss th ffts f tatmnt. If th tm ntainsny mat s, th an say ds nt .Hv, if a ag nm f immat s (pay

inding a ag pptin f tm-initiating s)a psnt in th tm samp, th an is iky ttn, and fth aggssiv tatmnt is aantd72.It has n shn that a high fqny f stm s inAMl at diagnsis pdits high minima sida dis-as aft thapy and p pgnsis75. In manma,Abcb5 is a mak f maignant manma-initiatings, and Abcb5+ tm s dttd in patintsith manma sh a pimitiv ma phntypand at psitivy ith inia manma p-gssin9. In ast an, cD44+ -spifi gnsindd many knn stm maks and -atd ith dasd patint sviva76. Fthm,

hmthapy tatmnt inasd th pntag fcD44+cD24/ tm s in patints ith astan77, nsistnt ith th ativ hmsistanf ths tm-initiating s.

Many tm-initiating s a thght t sistantt hmthaptis sh as paitax and dxiin,f vais asns inding thi qisnt sypifating nat78, th high xpssin v f ATP-inding asstt (Abc) dg pmps9,60,79, th intinsihigh vs f anti-apptti ms11, thi ativsistan t xidativ DNA damag, and thi ffi-iny f DNA pai8082. Athgh tm-initiatings might mmny m sistant t thapythan th k f tms, th d vaiatin insnsitivity t thapis amng tm-initiating s,as has n shn f nma stm s21,83.

Simia t taditina antian dgs, many nvtm-tagtd agnts as dsignd t tagtapidy pifating an s, s many tm-initiating s might as ativy insnsitiv tths agnts. F xamp, imatini (Gv; Nvatis)

tagts and inhiits th bcrAbl kinas, hih is thfsin ptin pdt f a hmsma tansa-tin and is sggstd t at as a ma sith thatpmts pifatin and diffntiatin f mtip-tnt pgnits in cMl84. bcrAbl is qid fth sviva f pifating pgnit s, t nt thqisnt cMl stm s50,85,86. Thf, athgh thmtatins a thght t amat in tm-initiatings, thi fntina ffts a msty manifstd f-th dnstam in th tm hiahy, ading tnpasti pifatin f pimitiv pgnits. As ast, imatini iminats pifating, mmittd -kamia pgnits, t nt pimitiv, qisnt tm-initiating s, and mst patints a sti psitiv f thfsin gn tansipts aft tatmnt8789. Atatinsin Ikas famiy zin fing 1 (IKZF1) a thght tsyngiz ith bcrAbl t ind ymphasti -kamia and ntit t dg sistan and disaspgssin90. Athgh it is iky that sva fatsntit t th pm, it has n shn that thdg sistan and disas n that a assi-atd ith imatini tatmnt f cMl might aviddy tagting an ssntia stm maintnan path-ay invving Hdghg44. As disssd av, tha th an typs in hih tm-initiating sd pifativ, and gth signaing pathaysa iky t hav imptant s in ths s. Ths

pathays a thf pmising thapti tagtsin sh ans.

If a patint has a ag nm f tm-initiatings and ny a sma nm f sh s a qidt gnat a tm, thn thapy has t highyffiint at kiing ths s t avid aps91. A th-apy that kis 95% f s in a tm might nsid-d ffiais asd n tm shinkag, t maya f th sviva f sffiint tm-initiatings t as vnta aps. Ths, vn if tm-initiating s a n m sistant t thapy than ktm s, thy an sti th ky t th imitatinsf tatmnt.

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6/18

If th an stm hypthsis is idy appia, itpay xtnds t npasms that a amna t ,sh as tain gm npasms (that is,tstiasminma) and nastmas. Indd, hmthapytatmnts f sh tms ftn iminat th ndif-fntiatd an s and pd sida masss thata nign tms mpsd f diffntiatd s92.This sggsts that thi stm mpnnts might inhnty snsitiv t hmthaptis and na tadapt t ntat thm. Sva nt stdis sggstdthat th sviva nfit f tastzma (Hptin;Gnnth/rh) a mnna antidy spifif pt tysin ptin kinaserbb2 (as knnas Her2) might at t its aiity t tagt asttm-initiating s93,94. Hv, mst patints ithmtastati ast an sti dvp sistan ithin1 ya f tastzma tatmnt 95, hih impis thgnti and/ pignti pastiity f ths tm-initiating s pmit thm t vv as a fntin ftm pgssin and/ thapti hangs.Tagting tm-initiating s might nt avid th

sam pms that hav n nntd f dadsin tating k tm ppatins: th mgnf dg sistan and th stin f inasingyfaty typs96. Nvthss, ndstanding thma asis f tm-initiating havi ia f th dsign f n statgis, inding mi-natin thapis t nt dg sistan.

Therapeutic opportunities

Th an stm hypthsis pvids a atina fsva thapti statgis ynd taditina anti-pifativ agnts (fIG. 1). Ptntia appahs t kitm-initiating s ind king ssntia sf-na signaing, inhiiting th sviva mhanismsf ths s, tagting tm-initiating sfamaks thgh antidy-asd yttxi appahs.Anth statgy is t ind tm diffntiatin,hih an ptntiay ahivd y inhiiting dvp-mnta pathays pignti pgamms. As manytm-initiating s might dpnd n a nih t main-tain thi idntity, tagting th nih d a statgy tindity inhiit diffntiat tm-initiating s.

Developmental pathways in self-renewal and differen-

tiation.As many tm s a thght t hav anaant diffntiatin pgamm and dgatdsf-na d a ky fat in many typs f

an38,45, sva dvpmnta signaing pathayshav m th nt fs f dg disvy ffts(fIG. 2; TABLe 1). Ding myni dvpmnt, this nsida sstak tn ths pathays, ithidntifia signaing nts that gnat, iv andintgat sva pathays97,98. Tm-initiating sand thi nihs might simiay pat as signaingnts, in hih mtip dvpmnta pathays aativ and nvg t nt sf-na.

wnt signaing has n shn t qid fsf-na f tm-initiating s in sva an-s, inding cMl and sqams ainmas41,42.extaa wnt inhiits, inding th std

Fizzd-atd ptins (SFrPs) and Dikkpf ptins(DKKs), hih at at th sfa t inhiit wnt sig-naing thgh its pts, hav n disvd99.Ativ divativs f ths antagnists, if dsignd t havdsia phamakinti pptis, d dvpdint antitm agnts, patiay as thy might at sppss wnt signaing in an vn hn th gnsnding adnmats pypsis i ptin (APc) -atnin a mtatd100. Sma-m antagnists fth ngni tansiptin fat TcF-atnin p-tin mpx hav n ptd101,102. Antidis against

vais wnts103,104, Fizzd ptins and wnt p-t -dnsity ipptin pt-atd ptin5(lrP5)lrP6 a as ing xpd.

Inhiitin f th Hdghg signaing pathayis as a via thapti statgy, and antidisagainst Hdghg and sma-m inhiits f thHdghg pt Smthnd hmg (SMo)hav n idntifid105,106(TABLe 1). It has n ptdthat phamagia inhiits f Hdghg signaingdispay ffiay in vais anima mds, inding ths

f asa ainma, mdastma, sma-ng an and panati an106110. M nty,it as shn that inhiitin f Hdghg signaing kiscMl tm-initiating s, impais th ppagatinf bcrAbl-divn cMl and th gth f imatini-sistant ms and hman cMl44. Hv, thsagnts a nt vy fftiv and th sts vay amnga mds, paty as it is diffit t maintainHdghg pathay ativityin vitro d t diffntia-tin nd nvntina t nditins111,112 and/qimnts f th psn f stma s 63,64,113.Hdghg igand xpssd y tm s an asativat th Hdghg pathay in th tm stmaminvinmnt, istating a paain qimntf Hdghg signaing114. Sh hangs mpiatin vitro sning assays, as disssd .

Inhiitin f Nth xpssin y antisns niaid thngy th phamagia kad fth ptas -stas, hih avs Nth (fIG. 2),has stiking antinpasti ffts in Nth-xpssingtansfmd s in vitro and in xngaft mds115117.A -stas inhiit has n shn t ind gt diffntiatin and gss n adnmas inmi aying a mtatin f thApc gn118. Anth-stas inhiit dpts tm-initiating s inain tms119. Hv, th thapti ind f-stas inhiits is na, as f thi inhi-

itin f mtip Nth pathays and th pssifft n nma stm s. rnty, antidis thata stiv f th Nth igand Dta-ik 4 (Dll4) shn t inhiit tm gth y dgatingangignsis itht mh f th txiity atd t-stas inhiitin in anima mds120,121. Shstiv tagting f an individa Nth pathaymight pvid a via statgy f impaiing nihfntin. Antagnist antidis against individaNth pts a as ing xpd122. In additint dvpmnta signaing pathays, th signaingpathays d imptant f th sf-na fdiffnt tm-initiating s123,124.

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|

DKK

Normalstem cells

Cancer

Haematopoietic,epidermal and intestinal

Colon carcinoma, epidermal tumoursincluding breast tumour, leukaemia,squamous cell carcinoma andtumours of the digestive tract

Wnt

Wnt Hh Notch

SFRP

SMOantagonist

Notch

-secretaseinhibitor

Gli

GliTCFLEF

FzLRP56

Haematopoietic,neural and germline

Medulloblastoma, basal cellcarcinoma, tumours of thedigestive tract, prostate cancer,leukaemia and multiple myeloma

SMO

STK36

Haematopoietic,epidermal and intestinal

Leukaemia, breasttumours, brain tumoursand lung cancer

Hh

RBPJ

PTCH

SUFU AKT-Cat

GSK3

CK1

Axin

APC

-cat

Ligand

Th is ging vidn that wnt, Hdghg andNth signaing a iky t intat ith n anthand ith additina signas, sh as n mph gntiptins (bMPs) and gth fats that a pdd

y tm-initiating s, th k tm s thiminvinmnt125. Ths signas nvg t gn-at th distintiv fats f tm-initiating s,inding sf-na, pifatin, sviva and dif-fntiatin. It is sti na h vais signaingpathays intat t maintain sf-na ativity indiffnt ans, and hth inhiitin f an individapathay d diffntiat tm-initiating s,inhiit thi pifatin ki thm. Th sts ddpnd n th stats and intatin f diffnt n-gni pathays. F xamp, NoTcH1 gats thPTeNphsphinsitid 3-kinas (PI3K)AKT path-ay in T at ymphasti kamia (T-All)126.

This mans that s f -stas inhiits an isniky t hav a thapti fft in ass f T-Allith dtd PTENand nstittivy ativatd AKT126.T stivy ki tm-initiating s, it might

nssay t inhiit mtip pathays in many ass.cnvsy, tagting a spifi dvpmnta pathayt pmt diffntiatin d a m gna stat-gy f iminating tm-initiating s (fIG. 1). It hasn shn that bMPs d ind th diffntiatinf cD133+ giastma tm-initiating s pdm-inanty t astyt-ik s, hih makdy attnatdthi tm-fming aiity in a pinia md127.

Th gatin f sf-na mains pyndstd and d invv tansiptina ntksand pignti pgamms that gat hmatinassiiity. It has n shn that mixd-inagkamia (Mll) fsin ptins, hih mdat

Figure 2 | sa atwa tat at wa d ma tm dmt ad a

tamat. The Wnt signalling pathway is activated by the binding of Wnt ligands to their receptors Frizzled (Fz)

and low-density lipoprotein receptor-related protein 5 (LRP5) and LRP6, leading to the release of-catenin (-cat) fromthe degradation complex, which consists of adenomatous polyposis coli protein (APC), axis inhibition protein (axin),

glycogen synthase kinase 3 (GSK3)and casein kinase 1 (CK1). This facilitates the entry of-catenin into the nucleus,where it regulates target gene transcription through association with the transcription factor TCFLEF (lymphoid

enhancer binding factor). Soluble Frizzled-related protein (SFRP) and Dickkopf protein (DKK) are endogenous secreted

antagonists of Wnt signalling. Activation of the Hedgehog (Hh) signalling pathway is initiated by binding of a Hh ligand to

protein patched homolgue (PTCH). This lifts suppression of Smoothened homologue (SMO), activating a cascade that

leads to the translocation of glioma-associated oncogene homologue (Gli) into the nucleus and the activation of target

genes. Serinethreonine protein kinase 36 (STK36: also known as FU) and suppressor of fused homologue (SUFU) actdownstream of PTCH and SMO to regulate Gli. Cyclopamine is a potent antagonist of SMO, and has been used as a tool

compound to study Hh signalling pathways both in vitro and in vivo. The core Notch pathway is activated by interaction

between the Notch ligand (Delta-like or Jagged) on one cell with the Notch receptor on another cell, resulting in two

proteolytic cleavages of the receptor. This mediates the release of the Notch intracellular domain, which enters the

nucleus and interacts with transcription factors including recombination signal binding protein for immunoglobulin Jregion (RBPJ; also known as CBF1). It has been suggested that alternative pathways involving AKT exist downstream of

Notch activation126,177. Various -secretase inhibitors can inhibit Notch cleavage and activation.

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8/18

hmatin stt thgh histn mdifiatin, anpgamm diffntiatd myid s and ativatsf-na in s ith n inhnt sf-na pp-tis46,128. Pym gp ptins, sh as nhan fzst hmg 2 (eZH2), a ssntia mpnnts yhih stm s vsiy pss gns that a atdt diffntiatin129. rnty, it as ptd that hyp-mthyatin f th gn nding th bMP pt 1bin a sst f giastma-initiating s is inkd tth ativity f eZH2 (Ref. 130). eZH2 as affts bMI1-mdiatd sppssin f thp16Ink4ap19Arf s t avtgth ast apptsis f stm s131. Invasiv asa

styp ast ans signifianty vxpss eZH2,hih d ad t dngatin f ast antyp 1 ssptiiity ptin (brcA1)132. Th pssiinvvmnt f ths pignti pgamms in diffnttm-initiating s and thi ptntia as thaptitagts a aas f intns stdy.

Survival mechanisms in tumour-initiating cells.Athgh kn itt at sviva pathays f tm-initiatings in vais tm typs, thy a ptntia tagts fkiing tm-initiating s. ctain ngni path-ays that a distint fm dvpmnta pathays mighthav a in th sviva f sm tm-initiating s,

t hav n fft n diffntiatin mtiptny.In AMl, na fat b (NF-b) as fnd t nstittivy ativ in pimitiv AMl s (hih ansidd kamia-initiating s), t nt in nmaHScs123. MG-132, a ptasm inhiit ith ptntNF-b signaing inhiity ativity, as shn t indapid dath in cD34+cD38 kamia-initiatings, t nt nma cD34+cD38 s. An intkin-4(Il-4)-spifi antidy dd th viaiity f thcD133 and cD133+ n an s and inasdth ffiay f hmthapy, sggsting that mapathays that ntit t k tm gth an as

sssfy tagtd t snsitiz tm-initiatings t yttxi thapis133. In additin, ativatin fth PTeNmammaian tagt f apamyin (mTor)signa tansd and tansiptin ativat 3 (STAT3)pathay in ast tm-initiating s is qid fthi viaiity and maintnan134, and th PI3K pathaygats sviva f tm-initiating s that sidin th pivasa nih f mdastma70. rntstdis as sggst an imptant f erbb2 inmaintaining tm-initiating s in ast an93,94,in additin t its psmd in k tm s. It isna h pathays that gat sf-na intatith ths that gat th sviva f tm-initiating

Table 1 | Selected agents targeting self-renewal signalling or tumour-initiating cell surface markers

Tat tat atwa

At ca dat stat r

Hedgehogpathway

5E1, a Sonic Hedgehogneutralizing mAb

Oesophageal and prostatecancer and medulloblastoma

Target validation 105

Cyclopamine, a SMO antagonist Solid tumours Target validation 38

Basal cell carcinoma Phase I 200

GDC-0449, a SMO antagonist Basal cell carcinoma andmetastatic colorectal cancer

Phase II Genentechwebsite(www.gene.com)

IPI-926 Advanced and/or metastaticsolid tumours

Phase I Infinity Pharmaceuticals website(www.ipi.com)

Wnt pathway A WNT2-specific mAb Melanoma and NSCLC Target validation 103,104

Various TCFb-catenin inhibitors Colon cancer Target validation 101,102

Notch pathway DBZ, a -secretase inhibitor Intestinal adenomas Target validationPreclinical

118

MK-0752, a -secretase inhibitor T-ALL and metastatic oradvanced breast cancer

Phase I Merck website(www.merck.com)

DLL4-specific mAbs Solid tumours Preclinical 120,121

ABCB5 3C2-1D12, an ABCB5-specific mAb Malignant melanoma Target validation 9

CD44 H90, a CD44-specific mAb Acute myeloid leukaemia Preclinical 148

P245, a CD44-specific mAb Breast cancer Preclinical 201

EPCAM Adecatumumab, anEPCAM-specific human mAb

Metastatic breast cancer Phase II Micromet website(www.micromet.de)

Edrecolomab, an EPCAM-specificmAb

Colon cancer Phase IIIII 142

Catumaxomab, a trifunctionalantibody against EPCAM and CD3

Malignant ascites and ovarianand gastric cancer

Phase IIIII Trion Pharma website(www.trionpharma.de)

MT110, a bispecific single-chainantibody against EPCAM and CD3

Lung and gastrointestinal cancer Phase I Micromet website(www.micromet.de)

ABCB5, ATP-binding cassette sub-family Bmember 5; DLL4, Delta-like 4; EPCAM, epithelial cell adhesion molecule; mAb, monoclonal antibody; NSCLC, non-small-celllung cancer; SMO, Smoothened homologue;T-ALL, T cell acute lymphoblastic leukaemia; TCF, T-cell factor.

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9/18

s, athgh it as nty shn that NoTcH1gats th PTeNPI3KAKT pathay in T-All126 anderbb2 xpssin in ast an94.

In giastmas, DNA damag spnss shnt pfntiay ativatd in gima-initiating sf and aft adiatin. Th adisistan f cD133+gima-initiating s an vsd ith a spifiinhiit f hkpint kinas 1 (cHK1) and cHK2(Ref. 81). In ast an, tain tm-initiating shav vs f ativ xygn spis (roS) thanspnding nn-tmigni s. Phamagiadptin f roS savngs in tm-initiating smakdy inass DNA damag and sts in adi-snsitizatin82. In additin, y stitin thghth xpssin f yin-dpndnt kinas inhiit 1A(cDNK1A; as knn as p21) imits DNA damagand maintains th sf-na f kamia-initiatings135. Ths piminay stdis highight th ptntiaf inhiiting DNA damag spnss136 t vm thsistan f tm-initiating s t thapy.

Cell surface markers and niche interaction.Maks nth sfa f tm-initiating s a as imptanttagts, patiay f antidy-asd thaptis(TABLe 1). Th sfa maks cD34, cD44, cD133and Abcb5 hav n sd t idntify tm-initiatings fm vais tm typs39. Th sfa maksmight ft th a igin and histy f thatpatia tm-initiating , and d highy

vaia amng tm typs, patints and vn ithinth sam patint. uniqy amng sh maks, cD44d hav sva s in tmignsis; its xps-sin is indd y ngni signas sh as -atninTcF4 and rasrafxtaa signa-gatd kinas(erK) pathays and ngativy gatd y th tmsppss p53 (Refs 137,138). Mst f ths maks axpssd in nma s, and s finding a thaptiind d a hang. Hv, th sm t sm phntypi diffns tn kamia-initiating s and HScs, inding diffns in thxpssin f THY1 mman gyptin, KIT andIl-3 pt- (Il3rA)139141. In patia, Il3rA,hih is nt psnt n nma stm s, is a spifisfa mak f AMl-initiating s141.

In additin t inding t thi tagts and inhiitingtagt-dpndnt signaing, mst thapti mn-na antidis (mAs) intat ith mpnntsf th immn systm thgh antidy-dpndnt

a yttxiity (ADcc) and/ mpmnt-dpndnt yttxiity, and immn spnss might pat f thi antitm mhanisms142. Intstingy,systmi administatin f a mA ditd againstAbcb5, hih inds ADcc in Abcb5+ maignant-manma-initiating s, xts tm-inhiityffts in a manma xngaft md9. Sm tm-initiating s xpss immn-tan maks143,ptntiay making thm sistant t immn attak.Hv, th antitm ativity f ths antidisan nhand y a yttxi immnnjgat ngind antidy inding t th tm andimmn s144,145, hih might a t ypass

vais immn tan mhanisms. Athgh Abctanspt ativity an st in tm-initiating sing ativy sistant t many nvntina tha-pis, it may mak th s sspti t atnativstatgis that tagt s ith fftiv dg ffx 146.

Agnts that tagt maks n th sfa f tm-initiating s d as k y affting th nih(fIG. 1). endsta (stasti) nihs hav n idnti-fid f th nma HScs63 and AMl-initiating s147.A mA spifi f th adhsin m cD44 asshn t adiat hman AMl-initiating s in vivoy king th taffiking f kamia-initiating st spptiv minvinmnts, and y ating thistm fat thgh diffntiatin148. endthianihs as vad f HScs149, and a a pssi-iity f kamia-initiating s. Na stm sa thght t aiz t vasa nihs 150152, andain tm-initiating s sid in a pivasanih69,70. Mv, stm -ik gima s havn shn t pmt tm angignsis71, sg-gsting that ths s might hp t at thi n

nih. Ths data indiat that signaing in nih int-atins an iditina, ith tm-initiatings as th th s and th tagt.

It is imptant t mphasiz that mh f th nih-intatin data has n taind fm anima mds,and th f th nih in vais hman tisss andans is nt yt a. Nvthss, th assiatintn tm-initiating s and th vasatds ais th intiging pssiiity that anti-angignithapy may k in pat y affting th vasa nihf tm-initiating s, and th d many a-sns t min agnts that tagt tm-initiating sith anti-angigni thapy. Statgis that spifiaytagt th nihs f tm-initiating s an as nvisind.

Therapeutic windows and combination strategies

Th ptntia thapti ind is aays a nnf any antian appah, inding ths that tagttm-initiating s. Hv, tm-initiating sthat xhiit vativ sf na might m snsi-tiv t agnts that inhiit sf-na pathays thannma stm s, th sf-na ativity f hihdpnds n th dvpmnta stag and tiss hm-stasis, amng th things. As mind ss f taintm sppss gns in pgnit s an ad tmaignant s ith inasd sf-na ativity47, th

might mhanisti diffns tn tm-initiating s and nma stm s ith spt tsf na. Knkt f th PTENtm spps-s ass th gnatin f tanspanta kamia-initiating s and th dptin f nma HScs inmi153,154. Intstingy, apamyin nt ny dptdkamia-initiating s t as std nma HScvs in this md. Athgh it has nt yt n n-fimd in th as f hman an, this finding high-ights th ptntia fasiiity f ahiving a thaptiind in tagting tm-initiating s. rnty,wnt-atnin signaing as shn t invvd inth maintnan f tans tm-initiating s

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10/18

and maignant hman sqams ainmas42,has wnt-atnin signaing is nt ssntia fnma pidma hmstasis. As disssd ,th SMo antagnist GDc-0449 as tatd inPhas I inia tias155, pviding piminay iniavidn f thapti inds f statgis that tagtsf-na signaing.

N that maks f tm-initiating s, indingIl3rA f AMl, a knn t xist141, th ntyimitd pti f ths that hav n idntifidi g as tm-initiating ppatins fm

vais systms a fth haatizd. As manytm-initiating s might iginat fm pgnits patiay diffntiatd s46,47, many f thma xptd t hav sfa maks distint fm thsf nma stm s. mAs that tagt diffnt pitpsf th sam tagt d hav diffnt thapti in-ds, ing t diffntia xps and signaing, asshn f cD44-spifi mAs148.

Anth xamp vant t th ptntia thaptiind f statgis tagting tm-initiating s is

th svatin that th minatin f a ptasminhiit (MG-132) ith th yttxi dg idaiininds apid and xtnsiv apptsis f th kamia-initiating ppatin hi aving nma HScs

via th in vitro and in vivo123. Th inhiitin f Nthsignaing y -stas inhiits is psmd t hava na thapti ind. Hv, tatmnt itha -stas inhiit smd t tt tatd inmi hn sing a psd dsing gimn156, sggst-ing that nma stm s d v qiky fmth tatmnt. Thf, asd n th phamakintipptis f th agnt, th thapti ind an impvd y ptimizing th dsing gimn, simia tth adjstmnt f dsing shd f yttxi agnts.

As disssd av, it is thght that sva pathays,inding ths that gat sf na and gth,nvg t gat tm-initiating s. If th is asffiinty ag thapti ind, tagting a mi-natin f pathays that a niqy ativ in tm-initiating s i m fftiv than inhiiting asing pathay. It has n ptd that simtansyking th Hdghg and pidma gth fatpt (eGFr) pathays sing ypamin and gfit-ini std in gth ast, apptsis and a das inth invasivnss f pstat an s157. Th PI3KAKTpathay has n ptd t gat Hdghg signa-ing in pat y nting ptin kinas A-mdiatd

gima-assiatd ngn hmg (Gi) ativity158,indiating th ptntia thapti va f miningHdghg antagnists ith PI3KAKT inhiits.

Many agnts that tagt tm-initiating s dif-fntiat ths s, and may nd t mind ithhmthaptis t iminat s fth dn thtm hiahy. Inhiitin f pmyyti ka-mia ptin (PMl) y asni tixid disptd thmaintnan f cMl-initiating s, indd th dif-fntiatin and pgssin thgh th y fths this qisnt tm s and snsitizdthm t p-apptti stimi159. In additin, th PI3Kpathay gats sviva f tm-initiating s that

sid in th pivasa nih fing adiatin inmdastma in vivo, and inhiitin f AKT signa-ing snsitizs ths s t adiatin-indd appt-sis70. Th PI3KAKT pathay gats AbcG2 ativityin gima-initiating s160, pviding anth atinaf mining PI3KAKT inhiits ith hmtha-ptis that a sstats f th Abc dg tanspt.

Givn th mging f Nth and Hdghg inth tm stma and vasat114,120,121, agnts ta-gting ths pathays might hav an impat n thtm-initiating s and tm vasat; thffts f ths agntsn dg divy shd thfas nsidd161. It is niva that hangingtm-initiating s ith th tagtd agnts andnvntina hmthapy adithapy d ntny m fftiv in kiing, t as day thdvpmnt f dg sistan mpad ith ithagnt an; hv, pinia vidn f this is ntyt avaia.

It is imptant t vaat diffnt agnts and mi-natins in th ntxt f th tm hiahy and ith

imaks162(fIG. 3b), t dtmin thi ffiay andt stdy th mgn f dg sistan in piniamds. It is as ia t stdy thi ffts n stmas and th vasat, and t f sy th th-apti ind f diffnt minatins. Hpfy, dsignd minatin statgis asd n datafm vant pinia mds an vm pathaydndany, nt dg sistan and hp t ahivng-tm missin in a inia stting.

Drug discovery with tumour-initiating cells

Th nvntina appah f antian dg disvyis t tagt pifatin ath than sf naand/ diffntiatin, and s is ftn iasd t sttagts ith hmgns xpssin pattns andptnt mpnds that ki th k tm s. Inadditin, sm taditina pinia mds may ntft inia mpxitis sh as tm hiahy.Tm-initiating s that dpnd n a nih anddvpmnta pathays invving paain jxta-in signaing may dmand m sphistiatd dgdisvy patfms than th t-dimnsina tisst stans xngaft mds that havtaditinay n sd t haatiz atnmstm s and atin signaing in an. Thag dy f vidn in sppt f th an stm hypthsis and th atd thapti statgis qi

adjstmnts t antian dg disvy patfms tmak thm m iniay vant (fIG. 3).

Tumour-initiating cell enrichment andin vitro cultureconditions.Ss fm hih t isat tm-initiatings ind samps fm patints ith pimay an,pimay tm xngafts and tain an ins(fIG. 3). Intstingy, it as svd that, as ith pimaytm s, sm an ins td nd nvn-tina nditins hav a tm hiahy asd n sta-ishd tm-initiating maks10,60,163165. Athghthi van t tm-initiating s fm pimaypatint samps sti qis fth haatizatin,

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|

Tissuedissociation

Primary xenograftsHuman tumours Cell lines

TIC populationknown?

Enrichment of TIC population: Surface or surrogate markers Self-renewal activities Optimized TIC culture

Monitor TIC population: Surface markers Fluorescent reporters Enzymatic reporters Functional tests

Monitor TIC activity and population in vivo: Tumorigenicity Cell surface markers Fluorescent reporters Enzymatic reporters

Enrichmentfor TICs

Standard end points Proliferation Survival Colony size

Standard end points Tumour volume

or size Metastasis Survival

TIC-related end points Relapse afterchemotherapytreatment

Minimal residualdisease

TIC-focused end points Tumorigenic activity

of enriched TICs TIC, lineage and

EMT markers Imaging TICs with

reporters orbiosensors

TIC-focused end points Differentiation markers

TIC Lineage EMT Self-renewal (for example,colony number)

Identify global and TIC-specific drug effects

Yes

No

Identify TIC population

Two-dimensionalculture

+ drugs

+ drugs+ drugs + drugsPlastic orcoatedplate

Matrixgrowth factors

Three-dimensionalorganotypic culture

+ drugs

Semi-solidmedia

Co-culturewith stromalcells

Replate

a

Tissuedissociationor fragment

Primary xenografts

Co-injection models

Human stromal cells,susceptible tomolecular engineering

Orthotopic models

Physiologicalmicroenvironment

Subcutaneous models

Simple, high-throughputassays

Human tumours Cell linesb

Figure 3 | Ata d d atm t tat tm-tat . Sources of tumour-initiating cells (TICs)

for enrichment and characterization include samples from cancer patients, primary tumour xenografts and certain cancer

cell lines that maintain tumour hierarchy. a |For in vitro studies, enrichment for TICs is likely to be a frequent but not a

necessary step in TIC characterization. TICs can be studied in heterogeneous systems provided their activities can be

monitored by cell surface markers or signalling reporter activities. Even highly purified TICs are expected to gradually

become more differentiated under tissue culture conditions. TICs that are dependent on a niche and paracrine or juxtacrine

signalling might be better maintained in three-dimensional organotypic culture than two-dimensional culture. Drug effects

on TICs can be studied according to standard end points (such as proliferation, survival and colony size) and TIC-focused

end points such as aberrant differentiation (for example, disproportionate presence of markers of TICs, lineage or

epithelialmesenchymal transition (EMT)) and self renewal. One way to measure self renewal in vitro is to track long-termcolony-initiating cells in colony-forming unit assays by replating in semi-solid media. TICs are defined by in vivo

experiments, and in vitro results always need to be validated in vivo. b | Depending on the life span of the bulk tumour, the

nature of the tumour hierarchy and the niche requirement for that particular tumour, different in vivo models and end points

may be required to study the drug effects on various TICs. For advanced tumours with a large TIC burden, traditional models

with standard end points (for example, tumour regression and metastasis) might be sufficient, whereas for other tumours,

it might be necessary to focus on the tumour-initiating activity through administration of the agents before and following

implantation of enriched or isolated TICs. TIC-related end points also include the effect on relapse after chemotherapeutic

treatment or the ability of residual tumour cells after treatment to re-engraft in in vivo xenograft models. To understand the

effect of the drug on tumour hierarchy, immunochemistry using antibodies against TIC, lineage and EMT markers needs to

be performed. To promote the tumourhost interaction, orthotopic models, subrenal capsule implantation and

subcutaneous co-injection models that mix tumour cells with stromal cells have been explored. It is essential to develop

in vivo imaging capacity to track TICs and to visualize the compartments in which they reside, and some preliminary

progress has been made in this area.

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th finding that tain an ins may ntain asppatin f tm-initiating s is imptant: ins an pvid sffiint matia f xtnsivma and signaing pfiing f ths s.

Th a sva ays t nih f tm-initiatings: sing sting thniqs t st f mi-natins f -sfa maks, sting t st f asppatin f s that ffx dys166,167, vaissm-f stm t nditins111,168,169(fIG. 3a).Athgh sting f sfa maks is s-f f isatin f tm-initiating s that xpssstaishd maks81,123, it d hanging ts this thniq in many th tms ing t aak f maks and/ th qimnt f mtipmaks15.

As th xpssin f th gns nding Abc dgtanspts sh as AbcG2 is a nsvd fat fsva stm ppatins fm a ang f ss, thffx f th DNA-inding dy Hhst 33342 has nsd t idntify and nih f tain typs f tm-initiating s160,166,167. Thtiay, this mthd may

m sita t nih fptntia tm-initiatings as it is ss nstaind y tiss spifiity thanth sfa maks disssd av. Hv, thkintis f dy xsin, a ptntiay sma ind fdtting stm s that xd th dy and th txiityf vais dys might imit fntina anaysis f thnihd ppatins. It is as pssi t nih ftm-initiating s y sting th sgat fn-tina pptis f stm s, sh as adhyd dhyd-gnas ativity165,170 and hmthapti sistan60.

Sm-f nn-adhnt t has n shnt nih fand ppagat sva typs f tm-initiating s, inding ths fm ain, ast andn an7,73,111,168,171. Th sm-f sph tas dvpd hn it as svd that nta nv-s systm (cNS) s gn n nn-adhnt sfasfm sphid nis (nsphs) that hav thapaity f sf na and an gnat a f th pin-ipa typs f th ain (that is, nns, astytsand igdndyts)172. cnvsy, s in sm-fadhnt t an as maintain stm -ik pp-tis if th pats sd hav a niq sfa (f xamp,inpating vais nitgn-ntaining fntinagps), a atd ith an apppiat matix and/ igand111,169,173.

It is ia t dvp t mthds and n-ditins that simat th gth and diffntiatin-

inhiity signaing that is pvidd y th nih,patiay f ting s fm pimay tms.Sm-f t nditins that hav n sta-ishd f nma stm s154,172,174 pvid sfstating pints. A n gnatin f high-thghptpatfms, sh as mifaiatd aays f xta-a matix th ms that a invvd inpaain and jxtain signaing, an sd tidntify vant minvinmnta signas f diff-nt tm-initiating s175. Sssf appahs tsstaining and xpanding nma stm s in sm-f t hav indd stimatin f th wnt,Hdghg and/ Nth pathays and inhiitin f

th bMP pathay174,176,177. Apppiat gth fatsmst as pvidd111,154,174 and fava mi-nvinmnts, sh as aminin-atd pats am-inin-ih matig, d nssay hpf111,169,174.Mtai ativity and xygn tnsin a th vaia-s t nsid in th t f stm s and tm-initiating s178,179.

As as nsiding sita sning nd pints,ffts t adapt ths stm t systms ttm-initiating s mst tak int ant th distintigins and haatistis f ths s. Dvpmntapathays that gat sf-na in t may aspvid thapti tagts, s th aan f xgnsfats may ia f tain sns. unik n-ma stm s, tm-initiating s mmny aymtatins that at thi gth fat dpndn spnss. Fth diffns in t qimntsmay xptd hn th igin haatistis fa tm-initiating is pgnit-ik. Finay, thmtatina and pathay pfis f tm-initiatings i vay ith tm styp and gad, and

s t nditins might hav t ptimizdadingy.

In vitro assays and screening methods.In many ass,inding nd sph nditins, in vitro t ftm-initiating s is xptd t pd mixdppatins f tm-initiating s and m dif-fntiatd pgny. This psnts th a hang(isating th fft f xpimnta intvntin ntm-initiating s) and an pptnity (th aiityt s diffntiatin as an nd pint). Mniting stm maks ith immnfsn fsntpt gn xpssin is amna t high-thgh-pt anaysis, and th adts hav n sssfysd t sn f nv gats f sf-na inmyni stm (eS) s180,181. Diffntiatin mak-s hav as n sd as pts t sn f smams gns that div inhiit stm dif-fntiatin182,183. basd n sh ssss, imagingpatfms and th mak-asd sns f mda-ts f tm-initiating havi an adiynvisind (fIG. 3a). Nvthss, ffiinty and iaytaining qantitativ data fm imags psnts dsignhangs in tms f th data tin, data handingand imag pssing184. Th th hang is that fmaks and pt gns hav n staishd f

vais tm-initiating s and thi diffntiatd

pgny.utimaty, th mpxity f tmstma int-

atins and tmmatix intatins in vivo might m aaty pdd y tivating mixd ppatins in th-dimnsina gantypi tsthat an maintain vais aspts fin vivo tmhstintatins and might nih f tm-initiating s.A m sssf in vitro sning statgy might ts mixd th-dimnsina gantypi ts andqantify and tak tm-initiating s ithin thmsing vais maks it-in qantitativ fs-nt nzymati pts. on hang in sn-ing th-dimnsina ts is th pdtin f a

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nsistnt gantypi stt in a high-thghptfashin, t agggatin mthds that dvpdf ia pdtin f myid dis fm eSs185 an phaps adaptd. T qantify spifi ppatins in th-dimnsina t fmats, high-spd imaging systms and atmatd imag anaysismthds an mind186.

Pgss has n mad ith kamia-initiatings and cNS tm-initiating s in tms fin vitroassay nditins123,169,171, and it has m fasi tndt high-thghpt in vitro anayss t sahf mpnds that diffntiat ki ths s124,169.In additin, in-dpth kndg f th stm s andpgnit s fm hamatpiti and cNS systmsis ginning t a dit mpaisn f nma stms and tm-initiating s fm ths tisss123,124,169.evntay, simia assays f th tm-initiating si staishd and ptimizd.

Ida t nditins shd sppt an pifatin in vitro itht gntypi atatins andith th tntin f phntypi havi mst

imptantyin vivo tmigni aiity v passags111.Hv, vn sht-tm t an as diffns inth in vivo ppatin aiity f HScs that hav idnti-a sfa maks, mphasizing th atin that isndd in daing nsins fm in vitro sts21,187.Atnativy, sing gnti manipatins t ahiva staiizd msnhyma-ik stat that apts manytm-initiating pptis an a high-thgh-pt sning in vitro, hih has nty gnatd nvads against tm-initiating s188.

In vivo tumour models.T vaat agnts that spifiaytagt tm-initiating s (fIG. 3b), it may nssayt fs n th tm-initiating ativity thgh admin-istatin f th agnts f and fing impantatinf tmigni s; th fft n staishd tmsmight nt as damati as in nasnt tms andd tak ng9. Atnativ nd pints ind thfft n aps aft hmthapti tatmnt, thfft n mtastasis and th aiity f sida tms aft tatmnt t -ngaft in in vivo xngaftmds. Hv, th fqny f tm-initiatings in sid tms sms t sstantiay highthan that f kamia-initiating s in kamia3,5,and nt mathmatia anayss hav fth indiatdthat tm-initiating s in advand tms maynt as a sma fatin189. Mathmatia md-

ing pdits that if pgnits aqi sf-naaiity thn sf-ning s an m t dminata tm53. Agnts that tagt tm-initiating smight thf sh damati ativity in tain ta-ditina mds f advand tms that hav a agtm-initiating dn.

Th fntin f a tm-initiating may m fftivy assssd hn th is thtpiayngaftd4,5,190,191, and tm mtastasis t spifi gansan ftn pdd in an orthotopic modl(BOX 2). Inmany pithia tms, an eMT ss f diffntiatinis fqnty vidnt at th invading dg f th tmand is iky t mdiat a dtahmnt and vnta

mtastasis57. cs that a ndging an eMT dnivay th pss t mtastati tm-initiating s, and s eMT maks d sd asimaks f vaating agnts that tagt tm-initiating s in mtastasis mds. Dspit an inasingaanss f thtpi mds, f sm an typs(sh as n an), pfman f an thtpiinjtin an thniay diffit. In ths ass,atnativ appahs, inding stans mdsith s sspndd in matig ( mixd ith stmas) and xngaft mds fating sna apsimpantatin, hav n xpd6,7.

In vivo dg disvy sning dmands a p-di, st-fftiv systm. Tm-initiating mds invving xntanspantatin f pimay tms4,5,17 hav imitatins f mdim- high-thgh-pt assays d t th intinsi vaiatin tn tmsand patia hangs f sing fshy std mat-ia, t d a hi f tsting andidat agnts.An atnativ appah is t gnat an insthat a nihd ith tm-initiating s. A highy

maignant ast an in (SK-3d) as gnatdy sqntia in vivo passag in piiin-tatd NoDScID mi, taking advantag f th hmthapy sist-an f th tm-initiating s60. Th SK-3d inis nihd f s that dispay a th ptativ pptisf ast tm-initiating s13; mv, ths smtastasiz and a apa f sia tanspantatin60.M nty, gima-initiating ins that a divddity fm pimay maignant gimas sss-fy td and xpandd nd sm-f adhntt nditins169. Gnti manipatin f diff-ntiatin stats an as sd t pd an ndif-fntiatd, tmigni haat in an ins188.Ths ins d m iniay vant thannvntina an ins, athgh th van fany sts taind in ins nds t nfimdin pimay an s.

T addss th imitatins f ins and pimaytm s, pimay tm xngafts that hav npassagd in vivo ff a niq systm f th stdy ftm htgnity and hiahy in pinia mds.Fagmnts f sgiay std tm a impantddity int immnmpmisd mi (thtpi-ay stansy). Th sting xngafts apassagd t n animas and a thf maintaindxsivy in vivo192. Th a ahitt andhtgnity f a pimay tm xngaft sy

sm ths f th igina patint tm and am mpx than th spnding fats f ta-ditina in xngafts. Thf, pimay tmxngafts pd ith apppiat xpimntaanaysis ts nstitt a tata pinia md ffftivy vaating ad mpnds and dvpingdg minatinand imak statgis162.

In vivo biomarker and imaging studies.Givn thattm-initiating s psnt ny a sppatin fth s in a tm and thi xistn might dpnd na nih, it is dsia t tak thm and visaiz th m-patmnts in hih thy sid f, ding and aft

Orthotopic model

A ytm in which tumour cll

ar implantd at th it o th

organ o origin.

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tatmnt in vivo. A piminay ndstanding f thfft f xpimnta intvntin n tm hiahyand tmhst intatin in vivo an ahivd yimmnhisthmisty sing antidis against maksf tm-initiating s, eMT, inag diffntiatinand diffnt stma s (fIG. 3b). F tm mdsthat hav a ag tm-initiating dn, gnsignat and miaay anaysis an as pvidimaks f tm-initiating s. Thgh in vivoinag taking f td manma s that adiffntiay fhm-njgatd, -xngaftdAbcb5+ manma-initiating s and Abc5 spp-atins hav n assayd f thi ativ ntitin ttm gth, sf na and diffntiatin9, and thfft f tatmnt an in pinip simiay stdid.cnty, th a n vis paths y hih t psdit imaging, athgh th s f ad antidisagainst spifi tm-initiating maks mightff an nty pint. T vaat mtastasis mdiatd ytm-initiating s, nn-invasiv magnti snanimaging f magntiay ad tm-initiating s,

as has n sd f nma stm s193, d sf.It d as dsia t dvp sgat ptgns th isnss that i a f in vivo mn-iting f th ativitis f sf-na signaing andtm-initiating s194. Tagtd divy f a ptgn has aady ad th atins f nma HScs t idntifid and mnitd y h-anima iv imag-ing195. M maks and pt gns f tm stm-ik pptis nd t staishd.

Hpfy, th impvmnt and mdifiatin fantian dg disvy patfms in ight f th anstm hypthsis i impv th inia van fpinia assays and mds. Ths mds i nt nyhp s t ndstand h nt hmthaptiand tm-tagtd agnts afft diffnt vs f thtm hiahy, t as va nv agnts that tagttm-initiating s. In additin, pinia stdissing ths mds might pvid data that spptniq inia minatins and imak statgisf agnts that tagt tm-initiating s.

Clinical strategies and outlook

Many aspts f th aant diffntiatin that is assi-atd ith p pgnsis in an an st xpaindy th an stm hypthsis. This is ndsdy th fat that, in inia tias f advand ans,tm gssin ftn ds nt tansat int iniay

signifiant inass in patint sviva. effiay againstminima sida disas, mtastasis, dayd aps andtm-f sviva a xptd t at ith thmhanism-asd ativity f agnts that tagt tm-initiating s.

Givn that tm gssin might nt thmst vant ay nd pint, imaks f tm-initiating s in patints h a iving anthapy nd t dvpd. Hv, tansating thmaks that a sd t nih f tm-initiatings int inia imaks is nt nssaiy staight-fad. F xamp, th cD44+ cD24/xps-sin in patints ith ast an that is dttd y

immnhisthmisty ds nt at ith vnt-f va sviva196, sggsting that nt a cD44+cD24/

an s a tmigni and sfa maks mightnt as nsvd as as iginay thght. Hv,gn signats divd y mpaing cD44+ cD24/ast an s ith nma ast pithia s,hih might ft th pignti stat f th tms, a atd ith dasd patint sviva76,197.ciating tm s, athgh xtmy a, aa ptntia atnativ t invasiv ipsis as a sf tm tiss f th dttin, haatizatin andmniting f tm-initiating s fm sid tms.A mihip thngy that is asd n mifidisas shn t snsitiv and a t dtt iatingtm s in amst a f th xamind patints ithnt ainmas198.

It is as imptant t stdy th van t tm-initiating s f nt imaks, sh as pstat-spifi antign (PSA) f pstat an and min16 (Muc16; as knn as cA125) f vaian an,hih a ginning t gid inia tias and thapy.

bth PSA and Muc16 a xpssd in diffntiatdtm s; it is sti na hth thy a sgatny t a k tm ppatin t a tm-initiating ppatin as . If gnti (f xamp,gn ampifiatin) pignti (f xamp, pmtmthyatin) hangs mtip hangs f atdpathays d sd t pdit th dpndn f atm-initiating n tain ngni pathays,sm f th sf-na signaing ms dpsnt an Ahis H f an. Inhiits f thspathays d hav nsida antitm ativityan199. In this gad, a diagnsti pd t p-sn patints ith sh gnti pignti hangsmight ssntia f dg dvpmnt.

Amng vais agnts that tagt sf-na path-ays, sma ms that tagt th Hdghg pathaya in ay inia stdis, and hav shn pmisingsts (TABLe 1). Th SMo antagnist ypamin asshn t ad t apid gssin f asa ainmain a f patints in hih it as tstd200. In additin,an ay-administd sma-m antagnist fSMo, GDc-0449, has shn imitd txiity and patiaspnss in advand asa ainma tms in aPhas I inia tia. It is advaning t Phas II tias fmtastati ta an and th advand pith-ia tms155. Th majity f patints ith asa ainma hav gnti mtatins in Hdghg path-

ay mdiats38; it is na hth GDc-0449 i as fftiv in th tm typs that d nt havsh mtatins hth it mst mind ithth agnts t sh inia ativity. This minatinappah d fth mpiatd y th mging f Hdghg signaing in tm stma s114,161.GDc-0449 and th SMo antagnists (TABLe 1) ithf pvid an intsting tst f inia statgisin tagting na signaing.

Th a sva antidis against sfamaks f tm-initiating s in inia stdis(TABLe 1). ePcAM is highy xpssd in nmssid tms, and as nty shn t xpssd

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n tm-initiating s fm ast, pstat, nand panati an4,7,8. ePcAM-spifi mAs havshn a imitd ffiay in inia tias142, sggstingthat immn tan ADcc stimatd y thsmAs y itsf might nt fftiv in kiing ePcAM-vxpssing tm s in inia sttings. Tvm th imitatins f th nakd antidis, at-maxma as dsignd t ind t th hman ePcAM(th tagt n th tm) and hman cD3 (th tagtn T s), inging an s int pximity ith thimmn-systm s that an dsty thm. In addi-tin, atmaxma inds ADcc and is ndgingadvand stdy in patints ith maignant asits145.cnvsy, mAs against cD44 an diffntiattm-initiating s and hav sing-agnt ativity intain pinia mds148,201(TABLe 1). It mains t sn hth ths mAs i sh sing-agnt ativityin inia sttings hth thy i as nd t pd ith yttxi appahs.

Vais dg sning patfms that spifiaydsignd t tagt tm-initiating s hav gn t

idntify nv dg ads169,188. whth tm-initiating-tagtd thapis a fftiv as sing agnts, intms f sht- and ng-tm inia nd pints, ipay dpnd n th ifspan f th k tms and th symptms thy as. F tms inhih mst tm s a sht-ivd th tm-initiating dn is ag, ntind tm gthand maintnan may highy dpndnt n th ativityf tm-initiating s. Tms ith ppatinsf pifating pgnit-ik s (BOX 1) may tak ang tim t gss, vn if tm-initiating s adstyd. F sm ans, ntind athgh im-itd pifatin f k tm s might sffiintt as ivsi pathgia damag. It is nssay t

ki a an s that hav th ptntia t ntitt disas, patiay f ans ith itt vidnf hiahia ganizatins15. Gnti and pigntiinstaiity d as nfnd th fftivnss fagnts that this ffiinty tagt tm-initiatings. It may imptant t min agnts that tagttm-initiating s ith nvntina agnts thatd th k f th tm, and th ptima mannf minatin d dpnd n th thapti in-d f th agnts, th if span f th k tm s,and th nat and staiity f th tm hiahy inthat patia tm typ.

M k is qid t ndstand h nt,patiay fftiv, hmthapti and tm-tagtdagnts afft diffnt vs f th tm hiahy (sth fig in BOX 1). Sm minatin tatmnt stat-gis hav aady mgd fm pinia stdis, ashighightd in pvis stins. In th ft, mi-natin thapy, inding yttxi, tm-tagtddgs and agnts that tagt tm-initiating s, may aimd at tm-initiating s, apidy pifat-

ing tm s and thi nihs simtansy sqntiay(fIG. 1). It is hpd that this n statgy ist in th apid mva f a tm sppa-tins and avid th pssi ppatin f th tmmass y tm-initiating s y iginay diffn-tiatd tm s that hav gaind na ativity(s th fig in BOX 1). Athgh th paths f dv-ping agnts that tagt tm-initiating s a ntstaightfad, th an stm hypthsis pvidsan imptant famk f dg disvy and antatmnt, ith th ptntia t find nv antitmativitis, t hav an impat n ans ith ndiff-ntiatd phntyps and t yid ng-tm nfits fmany patints ith an.

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