LETTER. A restricted cell population propagates glioblastoma growth after chemotherapy

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1 oi:1.138/nture11287 A restrite ell popultion propgtes glioblstom growth fter hemotherpy Jin Chen 1, Ynjio Li 1, Tzong-Shiue Yu 1,2 {, Renée M. MKy 1, Dennis K. Burns 3, Steven G. Kernie 1,2 { & Luis F. Pr 1 Glioblstom multiforme is the most ommon primry mlignnt brin tumour, with mein survivl of bout one yer 1. This poor prognosis is ue to therpeuti resistne n tumour reurrene fter surgil removl. Preisely how reurrene ours is unknown. Using genetilly engineere mouse moel of gliom, here we ientify subset of enogenous tumour ells tht re the soure of new tumour ells fter the rug temozolomie () is ministere to trnsiently rrest tumour growth. A nestin-dtk- IRES-GFP (Nes-DTK-GFP) trnsgene tht lbels quiesent subventriulr zone ult neurl stem ells lso lbels subset of enogenous gliom tumour ells. On rrest of tumour ell prolifertion with, pulse-hse experiments emonstrte tumour re-growth ell hierrhy originting with the Nes-DTK-GFP trnsgene subpopultion. Abltion of the GFP 1 ells with hroni gnilovir ministrtion signifintly rreste tumour growth, n ombine n gnilovir tretment impee tumour evelopment. Thus, reltively quiesent subset of enogenous gliom ells, with properties similr to those propose for ner stem ells, is responsible for sustining long-term tumour growth through the proution of trnsient popultions of highly prolifertive ells. We hve extensively stuie series of mouse strins hrbouring onitionl lleles of the tumour suppressors Nf1, p53 (lso known s Trp53) n Pten, whih spontneously evelop mlignnt glioms with 1% penetrne, n hve ientifie the soure ells of these tumours s eriving from the subventriulr zone (SVZ) 2 4. Thus, we propose tht ult neurl stem ells (NSCs) re the likely soure of these tumours 2. We wishe to etermine whether Nes-DTK-GFP trnsgene 5, originlly evise to mrk ult NSCs, woul lso mrk enogenous gliom ells. Elements of the nestin gene n rive trnsgene expression speifilly in ult NSCs 2,5 (Fig. 1). The trnsgene lso hrbours ssette ontining moifie version of the herpes simplex virus (HSV) thymiine kinse (DTK), llowing for temporlly regulte bltion of iviing neurl progenitors by systemi gnilovir (GCV) ministrtion, n n IRES-GFP ssette to mrk Nes-DTK-expressing ells in the bsene of GCV. The Nes-DTK-GFP trnsgeni mie showe the expete expression in both glil fibrillry ii protein (GFAP)-positive ult NSCs n erly oubleortin (DCX)-positive neurl progenitor ells (NPCs) in the mjor ult NSC nihe: the SVZ of the lterl ventrile (Fig. 1b, ). To vlite tht the GCV-tivte Nes-DTK-GFP trnsgene oul effetively eliminte enogenous neurl stem/progenitor ells, 1-month-ol trnsgeni mie were trete with GCV for 2 weeks. In ontrol mie, the rostrl migrtory strem (RMS), forme by NPCs migrting from the SVZ to the olftory bulb, is visulize by Nissl stining. In ontrst, the RMS ws severely iminishe in Nes-DTK-GFP mie fter GCV tretment (Fig. 1b, bottom). Consistent with this observtion, DCX immunostining inite the bsene of NPCs in the SVZ of GCV-trete Nes-DTK-GFP nimls (Fig. 1, bottom). Beuse, in the presene of GCV, HSV TK trgets only proliferting ells, the GFAP 1 NSCs tht remine quiesent were unffete, lthough they lso express the trnsgene, s inite by GFP (Fig. 1, bottom). Thus, our Nes-DTK-GFP trnsgene is speifilly expresse in SVZ quiesent b WT; GCV Nes- Δ TK; PBS Nes- Δ TK; GCV f Ki67 + ells (%) ~5.6 kb rt nestin promoter HSV ΔTK IRES GFP Nestin 2n intron SVZ RMS 6 *** *** Overll GFP LV 2 μm GFP + Per ent survivl g 1 5 SVZ LV 5 μm P =.8 GFP DCX GFAP e Time (ys) Tumour 1 Tumour 2 Tumour 3 Tumour 4 Mut7 (n = 17) Mut7; Nes- ΔTK (n = 22) h Mut7 Mut7; Nes- Δ TK 5 μm 5 μm Figure 1 Chrteriztion of the Nes-DTK-GFP trnsgene., Digrm of the Nes-DTK-GFP trnsgene. b,, GCV ministrtion bltes NSCs in wiltype mie. b, Representtive Nissl stining of the SVZ region in wil-type mie trete with GCV (WT; GCV), Nes-DTK trnsgeni mie trete with PBS (Nes-DTK; PBS) n Nes-DTK trnsgeni mie trete with GCV (Nes-DTK; GCV); blk rrows inite the stem ell RMS, whih is gretly reue in Nes- DTK mie trete with GCV., GFP (trnsgene), GFAP (quiesent NSCs) n DCX (ommitte neurl progenitors) immunostining of the SVZ stem ell nihe. White rrowhes in mile pnel (Nes-DTK; PBS) inite DCX 1 GFP 2 ells more istl in the RMS. White rrowhes in bottom pnel (Nes- DTK; GCV) inite GFP 1 /GFAP 1 but DCX 2 quiesent NSCs., Representtive GFP immunostining in setions from two untrete glioms of Mut7;Nes-DTK mie. From tumour to tumour, vrying numbers of GFP 1 ells were observe. TOPO-3 stins nulei. e, Representtive GFP n Ki67 oimmunostining in two untrete glioms of Mut7;Nes-DTK mie. White rrowhes highlight GFP 1 but Ki67 2 ells, emonstrting tht mny trnsgene GFP 1 ells re quiesent. f, Perentge of Ki67 1 ells in GFP 1, GFP 2, n overll tumour popultion in glioms from untrete Mut7;Nes- DTK-GFP mie. g, Kpln Meier survivl urve of untrete Mut7 n Mut7;Nes-DTK nimls. No ifferene in per ent survivl ws observe. h, Representtive hemtoxylin n eosin stining of Mut7 n Mut7;Nes-DTK brins without tretment. Infiltrtive mlignnt glioms re present in the ortex (re rrowhe) of both genotypes. ***P,.1. 2 mm GFP TOPO-3m GFP Ki67 TOPO-3 1 Deprtment of Developmentl Biology & Kent Wlrep Center for Bsi Reserh on Nerve Growth n Regenertion, University of Texs Southwestern Meil Center, Dlls, Texs , USA. 2 Deprtment of Peitris, University of Texs Southwestern Meil Center, 5323 Hrry Hines Boulevr, Dlls, Texs 7539, USA. 3 Deprtment of Pthology, University of Texs Southwestern Meil Center, 5323 Hrry Hines Boulevr, Dlls, Texs 7539, USA. {Present ress: Deprtment of Pthology n Cell Biology, Columbi University, New York, New York 132, USA. 522 NATURE VOL AUGUST Mmilln Publishers Limite. All rights reserve

2 RESEARCH n proximl progenitor ells, n hroni GCV ministrtion effetively bloks neurogenesis by blting quiesent ells s they enter the ell yle. We bre the Nes-DTK-GFP trnsgene into hgfap- Cre;Nf1 fl/1 ;P53 fl/fl ;Pten fl/1 (Mut7) gliom-prone mie 3. Mut7 mie evelop mlignnt gliom, with full penetrne, by somti eletion of three of the most frequently mutte tumour suppressors in glioblstom multiforme: p53, NF1 n Pten 6. All Mut7;Nes-DTK-GFP mie lso evelope glioms n we observe only subset of tumour ells expressing GFP (Nes-DTK-GFP positive; Fig. 1, e). These ells lso o-expresse the neurl stem ell mrker Sox2 (not shown). We next exmine tumour ell prolifertion n foun tht Mut7;Nes-DTK- GFP tumours exhibite signifint proportion of Ki67 1 /GFP 2 ells, n onversely, the subset of GFP 1 tumour ells rrely o-stine for Ki67 (Fig. 1e, f). These t inite tht most trnsgene GFP 1 tumour ells re reltively quiesent in omprison to highly prolifertive (Ki67 1 ) subpopultion reminisent of the SVZ, where GFP 1 stem ells re quiesent ompre to GFP 2 progenitors (Fig. 1). Furthermore, s in wil-type mie, in whih the Nes-DTK-GFP trnsgene oes not ffet SVZ neurogenesis in the bsene of GCV ministrtion (Fig. 1b, ), introution of the trnsgene into the Mut7 geneti bkgroun i not ffet tumour evelopment or enhne survivl (Fig. 1g). Like Mut7 mie, Mut7;Nes-DTK-GFP mie evelope mlignnt gliom with 1% penetrne n with similr kinetis (Fig. 1g, h). is DNA lkylting gent tht is urrently the primry hemotherpy ministere to glioblstom multiforme ptients 7 s it hs trnsient tumour growth-rrest properties. We foun tht erites prolifertive ells in the enogenous murine glioms. ws ministere over severl ys to tumour-bering mie followe by BrU injetion 2 hours fter finl tretment, n the mie were kille 2 hours therefter (Fig. 2). The -trete mie showe mrke reution of BrU inorportion in both tumours n NSC nihes (Fig. 2b, ). Similr to trete gliom ptients fter tretment, the murine tumours reinitite ell ivision n growth. Thus, trgets proliferting ells but tumour reurrene is inevitble. To exmine the etils of tumour reurrene, we tre the first wve of tumour ell prolifertion fter ompletion of the rug regimen in tumour-bering mie by pulse hse using the BrU nlogues ClU n IU. ClU n IU were injete 1 y n 3 ys, respetively, fter the finl injetion (Fig. 2). Given the vrible but reltively low proportion of GFP 1 ells in ll tumours (Fig. 1, e), we resone tht if ell prolifertion reinitite rnomly in tumour ells fter tretment, then the number of GFP 1 ells tht woul inorporte ClU n/or IU shoul be low to insignifint. However, if renewe tumour ell prolifertion ws hierrhil n erive from the GFP 1 ells, bise inorportion of nuleotie nlogues into the reltively smll GFP 1 ohort of tumour ells woul result. Exmintion of tumour setions with immunohistohemistry revele tht the lrge mjority of ells inorporting ClU n IU fter reltively short hses lso ontine GFP expression (Fig. 2e; GFP perentge in ClU 1 popultion , GFP perentge in IU 1 popultion ). Moreover, IU 1 ells retine both GFP n ClU, initing tht fter erition of mjority of preexisting proliferting tumour ells, the re-emergent popultion of proliferting ells erive from Nes-DTK-GFP-expressing ells n not from rnom tumour or other speifi subsets of ells (Fig. 2e; ClU perentge in IU 1 popultion ). When the seon, IU, pulse ws prolonge to 7-y hse fter the ClU pulse, most IU ells ouble-lbelle for ClU (85 6 7%) but lost GFP expression (Supplementry Fig. 1 ). Thus, s in the norml SVZ stem ell nihe, over time the GFP-expressing gliom ell popultion gives rise to ells tht progressively lose stem ell properties (tht is, nestin expression n reltive quiesene) n onomitntly shut own the Nes-DTK- GFP trnsgene (Supplementry Fig. 1, ). Using enogenous linege tring of renewe ell ivision within tumours, we hve ientifie the BrU + (%) DMSO BrU Perfusion *** ClU b e IU GFP IU Perfusion DMSO ClU Merge 1 μm 5 μm Figure 2 trgets proliferting erivtives but not the GFP 1 quiesent ell popultion., injetion shem. Mut7 mie were trete with for 5 ys, injete with BrU 2 h fter the finl tretment, n kille 2 h lter for BrU immunostining. b, Representtive GFAP/BrU o-stining of gliom from DMSO- or -trete mie shows mrke reution in the number of BrU 1 ells., Quntifition of the perentge of mximl BrU 1 ells in glioms from Mut7 mie trete with or without showe signifint erese in the -trete mie. Dt re men 6 s.e.m.; n 5 6 for eh tretment; ***P,.1, Stuent s t-test., e, Nes-DTK 1 ells re resistnt to n proue new tumour ells., Shem of tretment n short-term lbelling with BrU nlogues. Mut7;Nes-DTK mie were trete with for 5 ys n then injete with the BrU nlogues ClU n IU 1 n 3 ys fter the lst tretment, respetively. e, Representtive tumour setion illustrting tht repopulting tumour ells fter tretment express the Nes-DTK trnsgene (GFP 1 ); merge pnel, ClU-inorporting (rrowhe) or IU-inorporting (rrow) ells lso express GFP riven by the Nes-DTK trnsgene. (Perentge of GFP 1 ells in the ClU 1 popultion ; perentge of GFP 1 ells in the IU 1 popultion ; n 5 5.) Note tht the mjority of ClU 1 ells n IU 1 ells re positive for GFP expression, n lso tht the mjority of IU 1 ells re ClU 1, initing tht the ClU 1 ells gve rise to the IU 1 ells. reltively quiesent Nes-DTK-GFP-expressing tumour ells s the primry soure of proliferting n eventully non-iviing tumour ell erivtives (Supplementry Fig. 1, ). In ition, the t show tht in our enogenous gliom moels, trgets the proliferting erivtives but not the GFP 1 quiesent ells. One preition woul be tht erition of the GFP 1 ells in the enogenous tumours shoul onsierbly erese the emergene of new iviing tumour ells. We teste this preition t two stges: erly enogenous tumour evelopment n vne tumour evelopment. First, Mut7;Nes-DTK-GFP mie were trete with GCV beginning t 8 weeks of ge the erliest time of etetble pre-tumorigeni nomlies 3. To pture most quiesent GFP 1 ells in iviing stte, GCV tretment lste for 1 weeks, requiring three osmoti mini-pump plement surgeries tht unfortuntely were ompnie by omplitions, resulting in onsierble vrine of rug elivery. Drug-elivery effiy ws qulittively mesure by exmintion of the norml stem ell nihe response to rug (Fig. 1b, ) s mnifeste by resiul RMS (Supplementry Fig. 2). Aoringly, tumour iniene vrie from mouse to mouse, with some perishing GFAP BrU TOPO-3 23 AUGUST 212 VOL 488 NATURE Mmilln Publishers Limite. All rights reserve

3 RESEARCH LETTER erly with lrge tumours. However, s group, the GCV-trete ohort showe ler survivl vntge (Fig. 3). After 1 weeks of tretment, the only surviving mie were mong those trete with GCV (Supplementry Fig. 2b), n nlysis of their brins revele only lowgre lesions (Supplementry Fig. 2). When GCV ws effetively elivere, survivl ws substntilly prolonge n tumour progression ws severely impire. Thus, elimintion of Nes-DTK-GFP 1 ells t erly/pre-tumorigeni stges prevents evelopment of high-gre glioms. A seon GCV regimen ws strte in 1-week-ol tumour-bering mie. Most 1-week-ol Mut7 mie o not show neurologil symptoms, yet histologil exmintion of lrge ohorts showe tht ll mie hrbour stroytoms of iffering gres (Supplementry Fig. 3). We subjete 1-week-ol Mut7;Nes-DTK-GFP or Mut7 mie to GCV or sline for 2 months. GCV tretment of Mut7;Nes-DTK-GFP mie improve survivl by pproximtely 3 ys ompre to sline Per ent survivl week GCV tretment P = Dys post-gcv ΔMe sur. = 26 ys Mut7 GCV (n = 1) Sline (n = 8) + PBS b e f Mut7 g H&E Sox2 + PBS 2 μm Per ent survivl Control Mut7;Nes- Δ TK 1 5 P = Dys post-gcv ΔMe sur. = 31.5 ys 5 μm 1 week GCV tretment GCV (n = 7) Sline (n = 7) Nestin GFP DAPI 2 μm 1 Control Ki67 Ki67 + ells (%) Mut7 GCV (n = 5) Sline (n = 4) 5 P = Dys post-gcv ΔMe sur. = 1.5 ys n = 4 Control 2 mm ** n = 3 Figure 3 GCV tretment prolongs survivl of Mut7;Nes-DTK mie., Kpln Meier urve of Mut7;Nes-DTK mie trete with sline or GCV for 1 weeks strting t 8 weeks of ge showe ler survivl vntge for the GCV-trete mie (n 5 1 for GCV-trete; n 5 8 for sline-trete; P vlues etermine using log-rnk test). DMe sur., hnge in mein survivl. b, Kpln Meier survivl urves of Mut7;Nes-DTK (left) or Mut7 (right) mie trete with GCV or sline for 2 months strting t 1 weeks of ge. GCV tretment inrese survivl of Mut7;Nes-DTK mie ompre to sline tretment but h no suh effet on the Mut7 mie. n for the GCVtrete mie; n for the sline-trete mie; P vlues etermine using log-rnk test., GFP/nestin o-immunostining of glioms from ontrol (Mut7 mie trete with GCV or Mut7;Nes-DTK mie trete with PBS) n GCVtrete Mut7;Nes-DTK mie. Elimintion of GFP n nestin ouble-positive ells in the Mut7;Nes-DTK mie trete with GCV from 1 weeks., Representtive hemtoxylin n eosin stining of ontrol n GCV-trete Mut7;Nes-DTK brins. The tumours in the GCV-trete Mut7;Nes-DTK mie re less infiltrtive thn in ontrol. Tumours re inite by blk rrows. e, Representtive hemtoxylin n eosin (H&E) n Sox2 stining of tumour eges in ontrol n GCV-trete Mut7;Nes-DTK tumours showing the GCVtrete tumours hve efine bounry (otte line) n lk infiltrtive Sox2 1 ells. f, g, GCV tretment ereses prolifertion inex in Mut7;Nes- DTK tumours. f, Representtive Ki67 stining of ontrol (top) or GCV-trete Mut7;Nes-DTK tumours (bottom) showing the mrke erese in prolifertion in the GCV-trete Mut7;Nes-DTK tumours. g, Quntifition of the perentge of Ki67 1 ells in tumour regions with highest number of proliferting ells in ortex. The perentge is signifintly erese in GCVtrete Mut7;Nes-DTK mie (n 5 3) versus ontrol (n 5 4). Dt re men 6 s.e.m.; **P,.1, Stuent s t-test. ontrol, wheres neither tretment ws effetive in improving survivl of Mut7 mie (Fig. 3b). Consistent with this, GFP n enogenous nestin ouble immunostining showe tht GFP 1 ells were suessfully eliminte in glioms of Mut7;Nes-DTK-GFP mie fter prolonge GCV tretment (Fig. 3). The resiul tumours in these tumour-bering Mut7;Nes-DTK mie trete with GCV beginning t 1 weeks of ge i not hve the lssi gliom feture of invsiveness but inste were irumsribe with well-efine bounries (Fig. 3, e n Supplementry Fig. 2, e). Although the Mut7;Nes-DTK-GFP trnsgene is only expresse in subset of ells in the untrete tumour (Fig. 1e), tumours in GCVtrete Mut7;Nes-DTK mie showe mrke reution of Ki67 1 ells n stem ell mrkers (Fig. 3e g). These t further support the hypothesis tht hroni GCV ministrtion progressively bltes the reltively quiesent GFP 1 tumour ell popultion n the remining GFP 2 tumour ells eventully exhust their prolifertive n infiltrtive potentil. The HSV TK system hs been wiely use s metho to inue enogenous ell suiie. A potentilly onfouning phenomenon is the bystner effet, whereby HSV-TK-expressing ells not only ommit suiie in the presene of GCV, but n lso inue the eth of neighbouring non-tk-expressing ells 6. However, severl NSCspeifi HSV-TK-expressing trnsgeni mie hve been reporte n no bystner effet hs been esribe To exmine whether tumour evelopment ws ppreibly impire by the bystner effet, we turne to trnsplnttion ssys. We first etermine whether GCV tretment woul lso blunt Mut7;Nes-DTK-GFP tumour growth in trnsplnttion ssy. Primry glioms from Mut7;Nes-DTK-GFP n Mut7 mie were issoite n iretly injete subutneously into nue mie in the presene of ontinuous GCV or sline tretment (Supplementry Fig. 4). Neither GCV nor sline ffete the tumour growth of Mut7-erive ells (Supplementry Fig. 4b). In ontrst, similr to previous report 12, mie trnsplnte with Mut7;Nes-DTK tumour ells n then trete with GCV evelope signifintly smller tumours tht ppere to be poorly vsulrize ompre to sline-trete ontrols (Supplementry Fig. 4 f). Thus, the Mut7 tumour ells trnsplnt effiiently in immunoompromise mie n, s in the enogenous setting (Fig. 3), GCV tretment severely impirs tumour evelopment fter trnsplnttion. To test for the bystner effet, we trnsue primry Mut7 ells with lentivirus hrbouring either ontrol RFP ssette or n HSV TK ssette (Supplementry Fig. 5). Mixe rtios of the two ell popultions were injete subutneously into nue mie n llowe to see tumours for 4 weeks, fter whih GCV ws ministere for 2 weeks (Supplementry Fig. 5b). The t inite tht the presene of 1%, 2% or 5% initil rtio of TKexpressing tumour ells i not impir tumour evelopment of the non-tk-expressing ells in the presene of GCV (Supplementry Fig. 5, ). In the extreme se when equl numbers of TK 1 (1 5 )ntk 2 (1 5 ) ells were injete ompre to 1 5 TK 2 ells lone, tumour evelopment ws equivlent in both ohorts, emonstrting tht GCV toxiity to 5% of the tumour ells i not exten into the TK 2 tumour ell popultion (Supplementry Fig. 5, ). In our enogenous tumours, suh bystner effet woul require the reltively rre GFP 1 tumour-propgting ells to be wiely toxi in orer to hve onsierble prrine effet on tumour properties. Inste, these stuies inite tht GCV ministrtion oes not hve n ppreible effet on ells outsie those expressing the TK gene, onsistent with other reently reporte stuies We onlue tht erition of the Mut7;Nes-DTK-GFP enogenous tumour ells through GCV tretment effetively isrupts the ontinue proution of tumour ells, s preite from the preeing t. Despite effetive epletion of GFP-expressing tumour-propgting ells with GCV tretment, onsierble resiul tumour mss remine (Fig. 3). We therefore ttempte therpeuti strtegy to eliminte both the rpily proliferting tumour ells n the quiesent N AT U R E V O L AU G U S T Mmilln Publishers Limite. All rights reserve

4 RESEARCH GFP 1 ells by sequentilly ministering n GCV (/ GCV), respetively (Fig. 4, b). Initilly it seeme tht this regimen i not prolong survivl beyon tht of GCV tretment lone (Fig. 4). However, exmintion of the brins of the sequentilly trete mie revele only vestigil tumours in the orsl brin tht showe no trnsgene GFP expression, initing effetive epletion (Fig. 4). The ellulr ensity of the resiul /GCV-trete tumours ws lower thn tht of tumours t the beginning of tretment (1 weeks), initing sttistilly signifint reution in tumour bulk. This ontrst ws mrkely enhne when trete tumours were ompre to untrete ontrol tumours (Fig. 4e), whih mnifeste in spetrum of survivl time from weeks. Thus, the ombine tretment h rmti inhibitory effets on orsl tumour growth in these mie. We were puzzle tht /GCV tretment i not signifintly prolong survivl beyon the GCV-only trete mie, in whih resiul irumsribe tumour mss remine (Fig. 3, e n Supplementry Fig. 2, e). Anlysis of the /GCV-trete brins revele tht, espite the mrke inhibition of originl tumour growth, these mie evelope novel tumours in the ventrl brin region. Six out of seven /GCV-trete mie h tumours in the brinstem region, wheres glioms in untrete Mut7 mie n their remnnts in the suessfully trete mie re preominntly lote in the orsl/ mibrin (Supplementry Fig. 6). Exmintion of the /GCVresistnt hinbrin tumours revele high enogenous nestin protein but no GFP expression (Supplementry Fig. 6b, ). We lso serhe b GCV Per ent survivl 1 5 GCV Dys fter tretment Control untrete /GCV trete Combine (n = 9, Me sur. = 55 ) GCV (n = 7, Me sur. = 52.5 ) (, Me sur. = 3.5 ) PBS (n = 7, Me sur. = 21 ) e Cell number per mm 2 5 µm 12, 1, 8, 6, 4, 2, P =.5 n = 8 5 μm P =.4 P =.1 n = 4 Untrete tumour Pre-tretment GCV only GFP Nestin DAPI /GCV Mut7:Nes- ΔTK Figure 4 Combintion tretment of n GCV inhibits gliom progression in erebrum., b, Therpeuti shem trgeting both CSCs n their proliferting progeny. Mut7;Nes-DTK mie were trete with for 5 ys, followe 2 ys fter by GCV., Kpln Meier survivl urve of Mut7;Nes-DTK mie with ifferent tretments. GCV-trete (n 5 7; mein survivl (Me sur.) 5 55 ys) n /GCV-trete (n 5 9; mein survivl ys) Mut7;Nes-DTK mie h similr survivl vntge over trete (n 5 6; mein survivl ys) or PBS-trete (n 5 7; mein survivl 5 21 ys) mie. P vlues were etermine using log-rnk test., GFP n nestin ouble immunostining of vestigil tumours in /GCV-trete Mut7;Nes-DTK mie (right) versus tumour in ontrol (left) emonstrtes epletion of CSCs s eviene by lk of GFP expression. e, Mximl ell ensity in ortil tumours with ifferent tretment regimens. Untrete Mut7 mie (n 5 6) were use s ontrol. Tumour ensity of 1-week-ol nonsymptomti Mut7 mie (pre-tretment) (n 5 8) ws use s referene strting point. Cell ensity ws signifintly lower in the /GCV-trete Mut7;Nes-DTK mie (n 5 6) ompre to untrete tumours (P 5.5), ompre to tumours in pre-tretment mie (P 5.4), n ompre to tumours from mie trete with GCV only (n 5 4). Dt re men 6 s.e.m.; P 5.1, Stuent s t-test. rhive mteril n foun tht smll perentge of Mut7 brins hrboure both orsl n ventrl tumours tht we now ppreite were inepenently rising n not extensions of the orsl tumours. Apperne of ventrl tumours in the presene of GCV inites n intive Nes-DTK-GFP trnsgene, n iret exmintion of untrete Mut7;Nes-DTK-GFP mie emonstrte tht the Nes-DTK-GFP trnsgene ws inee silent (Supplementry Fig. 6b, e). We next exmine the expression of tumour mrkers in these hinbrin tumours. In ontrst to typil Mut7 or Mut7;Nes-DTK-GFP glioms, ll ventrl tumours in the /GCV group showe low levels of enogenous GFAP but reltively high levels of S1B (Supplementry Fig. 6 n t not shown). Seven out of eleven tumours in the ventrl n brin stem region of GCV-trete mie (GCV lone or n GCV) showe histopthologil fetures of oligoenrogliom (Supplementry Fig. 7 ), inluing high PDGFR- levels (Supplementry Fig. 7). This is in ontrst to the pure stroyti tumours typilly observe in this mouse moel in the bsene of /GCV. Thus, these t inite tht the ventrl tumours tht beome evient fter /GCV tretment re oligostroyti, inepenently rising, n istint from the orsl tumours. Suh tumours were reently esribe in Mut3 mie 13, n the soure ws reporte to be oligoenroglil progenitors. We re urrently further hrterizing these tumours. Our Nes-DTK-GFP trnsgene lbels SVZ stem ells n, fortuitously, speifi subset of glioblstom multiforme ells tht possesses mny fetures propose for ner stem ells (CSCs). The CSC hypothesis hols tht some tumours re ompose of hierrhil re of ells of whih only subset retins both selfrenewl n ifferentition pity 14. In this moel, only CSCs hve the pity to sustin tumour growth n re responsible for reurrene fter therpy fils. The urrent stnr for evluting whether soli tumours ontin CSCs is n ex vivo limiting ilution tumorigeni trnsplnttion ssy into immunoefiient nimls 14. However, ontroversy regring the presene n frequeny of soli tumour CSCs remins, probbly s refletion of the vribility tht ompnies suh ssys Our stuy ientifies puttive enogenous gliom stem ell lote t the pex of ellulr hierrhy in tumour mintenne n reurrene fter hemotherpy (Fig. 4). Continue evlution of these ells n their properties, inluing isoltion n geneti linege tring, my yiel importnt insights into novel therpeuti trgets for this intrtble isese. METHODS SUMMARY Mie. All mie were mintine on mixe 129SvJ/C57BL/6/B6CAB bkgroun. Mut7 n Mut7;Nes-DTK mie were obtine by rossing mle hgfap-cre;p53 fl/fl mie with femle Nf1 fl/fl ;P53 fl/fl ;Pten fl/fl ;Nes-DTK mie. Genotyping for Mut7 mie ws performe s reporte previously 3. In vivo hemil ministrtion. Stok ClU (Sigm) n BrU (Sigm) were issolve in PBS t onentrtion of 8.5 mg ml 21 n 1 mg ml 21, respetively mg ml 21 IU (MP Biomeils) solution ws me fresh eh time 19.Tolbel iviing ells, 5 ml kg 21 stok solution ws injete intrperitonelly eh time oring to the experimentl esign. GCV (Cytovene-IV, Rohe Phrmeutils) tretment ws performe s esribe 5. For initil hrteriztion of Nes-DTK-GFP mie, 1-month-ol Nes-DTK-GFP or ontrol mie were ministere GCV (3 mg kg ) or PBS vi osmoti mini-pump (Moel 22,.5 mlh 21,Alzet) for 2 weeks. For tretment strting from 8 or 1 weeks, 15 mg kg GCV or PBS ws elivere through osmoti mini-pump (Moel 24,.25 mlh 21, Alzet); pumps were surgilly remove n reple every 4 weeks bse on the experimentl requirement. (Sigm) ws issolve in DMSO n injete intrperitonelly t ose of 82.5 mg kg for 5 ys. For the ombintionl therpy group, mie were first trete with for 5 ys n then osmoti minipumps with GCV were implnte 2 ys fter the lst injetion. Histology n immunohistohemistry. Mie were perfuse n brins were proesse s esribe erlier 2. Prffin brin hemtoxylin n eosin setions (5 mm) were reviewe by J.C. n D.K.B. inepenently. Tumour type n gres were etermine by D.K.B. Fourteen-mirometre ryostt setions were use for GFP/ClU/IU stining following reporte methos 19. Primry ntiboies were 23 AUGUST 212 VOL 488 NATURE Mmilln Publishers Limite. All rights reserve

5 RESEARCH LETTER use ginst GFAP (DAKO, 1:2,), Olig2 (Millipore, 1:1,), Sox2 (Millipore, 1:5,), nestin (BD Biosienes, 1:2), CD44 (BD Biosienes, 1: 75), GFP (Rokln, 1:2, Aves Lb, 1:5), BrU/IU (BD Biosienes, 1:1), BrU/ ClU (AbD Serote,1:5), Ki67 (Novstr, 1:1,), PDGFR- (Snt Cruz, 1:2). Horserish-peroxise-bse Vetstin ABC Kit (Vetor Lbortories) or Cy2/Alex-488, C3/Alex555, Cy5-lbelle seonry ntiboies (Jkson Lbs, Invitrogen) were use to visulize the primry ntiboy stining. Full Methos n ny ssoite referenes re vilble in the online version of the pper. Reeive 26 Jnury 211; epte 7 June 212. Publishe online 1 August Chen, J., MKy, R. M. & Pr, L. F. Mlignnt gliom: lessons from genomis, mouse moels, n stem ells. Cell 149, (212). 2. Alntr Llguno, S. et l. Mlignnt stroytoms originte from neurl stem/ progenitor ells in somti tumor suppressor mouse moel. Cner Cell 15, (29). 3. Kwon, C. H. et l. Pten hploinsuffiieny elertes formtion of high gre stroytoms. Cner Res. 68, (28). 4. Zhu, Y. et l. Erly intivtion of p53 tumor suppressor gene ooperting withnf1 loss inues mlignnt stroytom. Cner Cell 8, (25). 5. Yu, T. S. et l. Trumti brin injury-inue hippompl neurogenesis requires tivtion of erly nestin-expressing progenitors. J. Neurosi. 28, (28). 6. Ishii-Morit, H. et l. Mehnism of bystner effet killing in the herpes simplex thymiine kinse gene therpy moel of ner tretment. Gene Ther. 4, (1997). 7. Stupp, R. et l. Riotherpy plus onomitnt n juvnt temozolomie for glioblstom. N. Engl. J. Me. 352, (25). 8. Gri, A. D. et l. GFAP-expressing progenitors re the prinipl soure of onstitutive neurogenesis in ult mouse forebrin. Nture Neurosi. 7, (24). 9. Deng, W. et l. Ault-born hippompl entte grnule ells unergoing mturtion moulte lerning n memory in the brin. J. Neurosi. 29, (29). 1. Singer, B. H. et l. Compenstory network hnges in the entte gyrus restore long-term potentition following bltion of neurogenesis in young-ult mie. Pro. Ntl A. Si. USA 18, (211). 11. Snyer, J. S. et l. Ault hippompl neurogenesis buffers stress responses n epressive behviour. Nture 476, (211). 12. Bo, S. et l. Stem ell-like gliom ells promote tumor ngiogenesis through vsulr enothelil growth ftor. Cner Res. 66, (26). 13. Liu, C. et l. Mosi nlysis with ouble mrkers revels tumor ell of origin in gliom. Cell 146, (211). 14. Clrke, M. F. et l. Cner stem ells perspetives on urrent sttus n future iretions: AACR Workshop on ner stem ells. Cner Res. 66, (26). 15. Boiko, A. D. et l. Humn melnom-inititing ells express neurl rest nerve growth ftor reeptor CD271. Nture 466, (21). 16. Ishizw, K. et l. Tumor-inititing ells re rre in mny humn tumors. Cell Stem Cell 7, (21). 17. Kelly, P. N. et l. Tumor growth nee not be riven by rre ner stem ells. Siene 317, 337 (27). 18. Quintn, E. et l. Effiient tumour formtion by single humn melnom ells. Nture 456, (28). 19. Veg, C. J. & Peterson, D. A. Stem ell prolifertive history in tissue revele by temporl hlogente thymiine nlog isrimintion. Nture Methos 2, (25). Supplementry Informtion is linke to the online version of the pper t Aknowlegements The uthors thnk S. MKinnon, A. Deshw, L. MClelln, S. Kenney n P. Leke for tehnil ssistne, n Pr lbortory members for helpful suggestions n isussion. ClU n IU preprtion n stining protool ws provie by D. A. Peterson t Roslin Frnklin University. This work ws supporte by grnts wre to S.G.K. (RO1 NS ) n to L.F.P. by the Golhirsh Fountion, the Jmes S. MDonnell Fountion (JSMF-22226), Cner Prevention Reserh Institute of Texs (RP 1782) n the Ntionl Institutes of Helth (R1 CA131313). L.F.P. is n Amerin Cner Soiety Reserh Professor. Author Contributions J.C. n Y.L. performe the experiments. T.-S.Y. n S.G.K. ontribute vitl regents. J.C. n L.F.P. esigne the experiments. J.C., R.M.M., D.K.B. n L.F.P. nlyse the t. J.C., R.M.M. n L.F.P. wrote the pper. Author Informtion Reprints n permissions informtion is vilble t The uthors elre no ompeting finnil interests. Reers re welome to omment on the online version of this rtile t Corresponene n requests for mterils shoul be resse to L.F.P. (luis.pr@utsouthwestern.eu) N AT U R E V O L AU G U S T Mmilln Publishers Limite. All rights reserve

6 RESEARCH METHODS Mie. All mouse experiments were pprove by n performe oring to the guielines of the Institutionl Animl Cre n Use Committee of the University of Texs Southwestern Meil Center t Dlls. All mie were mintine on mixe 129SvJ/C57BL/6/B6CAB bkgroun. Mut7 n Mut7;Nes-DTK mie were obtine by rossing mle hgfap-cre;p53 fl/fl mie with femle NF1 fl/fl ;P53 fl/fl ;Pten fl/fl ;Nes-DTK mie. Genotyping for Mut7 mie ws performe s reporte previously 3. In vivo hemil ministrtion. Stok ClU (Sigm) n BrU (Sigm) were issolve in PBS t onentrtion of 8.5 mg ml 21 n 1 mg ml 21, respetively mg ml 21 IU (MP Biomeils) solution ws me fresh eh time 19.Tolbel iviing ells, 5 ml kg 21 stok solution ws injete intrperitonelly eh time oring to the experimentl esign. GCV (Cytovene-IV, Rohe Phrmeutils) tretment ws performe s esribe 5. For initil hrteriztion of Nes-DTK-GFP mie, 1-month-ol Nes-DTK-GFP or ontrol mie were ministere GCV (3 mg kg ) or PBS vi osmoti mini-pump (Moel 22,.5 mlh 21,Alzet) for 2 weeks. For tretment strting from 8 or 1 weeks, 15 mg kg GCV or PBS ws elivere through osmoti mini-pump (Moel 24,.25 mlh 21, Alzet); pumps were surgilly remove n reple every 4 weeks bse on the experimentl requirement. (Sigm) ws issolve in DMSO n injete intrperitonelly t ose of 82.5 mg kg for 5 ys. For the ombintionl therpy group, mie were first trete with for 5 ys n then osmoti mini-pumps with GCV were implnte 2 ys fter the lst injetion. Histology n immunohistohemistry. Mie were perfuse n brins were proesse s esribe erlier 2. Prffin brin hemtoxylin n eosin setions (5 mm) were reviewe by J.C. n D.K.B. inepenently. Tumour type n gres were etermine by D.K.B. Fourteen-mirometre ryostt setions were use for GFP/ClU/IU stining following reporte methos 19. Primry ntiboies were use ginst GFAP (DAKO, 1:2,), Olig2 (Millipore, 1:1,), Sox2 (Millipore, 1:5,), nestin (BD Biosienes, 1:2), CD44 (BD Biosienes, 1: 75), GFP (Rokln, 1:2, Aves Lb, 1:5), BrU/IU (BD Biosienes, 1:1), BrU/ ClU (AbD Serote,1:5), Ki67 (Novstr, 1:1,), PDGFR- (Snt Cruz, 1:2). Horserish-peroxise-bse Vetstin ABC Kit (Vetor Lbortories) or Cy2/Alex-488, C3/Alex555, Cy5-lbelle seonry ntiboies (Jkson Lbs, Invitrogen) were use to visulize the primry ntiboy stining. Imges were tken using optil, fluoresene n onfol mirosopy (Olympus n Crl Zeiss) n ssemble in Aobe Illustrtor (Aobe Systems Inorporte)., BrU nlogues n pulse-hse experiments. To etermine effiieny, 1- to 11-week-ol Mut7 mie were first injete intrperitonelly with 82.5 mg kg for 5 ys. 5 mg kg 21 BrU ws injete 2 h fter the finl ministrtion n mie were perfuse 2 h fter BrU injetion. The brin ws then prffin-proesse n ut into 5-mm-thik slies. Hemtoxylin n eosin stining ws performe every 7 mm to ientify tumour lotion. Ajent tumour setions were selete for GFAP n BrU o-immunostining. For the short-term ClU hse experiments, 1- to 11-week-ol Mut7;Nes-DTK mie were first trete with for 5 ys. A totl of three oses of ClU were injete, with 2-h intervls, the y fter the finl injetion. A single ose of IU ws then injete 3 ys fter the finl injetion. For the long-term ClU hse experiments, 1- to 11-week-ol Mut7;Nes-DTK mie were first trete with for 5 ys. ClU ws injete 3 times y, with 2-h intervls, for 3 ys fter the finl injetion. A single ose of IU ws then injete 7 ys fter the finl injetion. Mie were perfuse 2 h fter the IU injetion n the brins ryoprotete in 3% surose, embee in OCT, n ut into 14-mm-thik frozen setions. GFAP n Ki67 o-immunostining ws performe every 14 mm to lote the tumour re. Ajent setions were selete for GFP/ClU/IU triple immunofluoresene stining. Quntifition. Beuse of the heterogeneous nture of the tumours, ell ensity, Ki67 inex n BrU 1 ell perentge were quntifie using the highest stining re 1. Briefly, stining ws heke uner low mgnifition n the highest stining re ws ientifie. The re ws viewe t 32 in three ontinuous 5-mm-thik setions n positive ells ounte using the mesure prmeters. For quntifition of GFP/ClU/IU triple stining, tumour res with t lest one ClU 1 ell were selete, n n 8 mm Z-stk imge ws snne n onstrute using onfol mirosopy (Olympus n Crl Zeiss). A totl of ten ifferent res within eh tumour ws imge n subjete to quntifition. 212 Mmilln Publishers Limite. All rights reserve