tumorigenic and metastatic activity of human thyroid cancer … · 2013. 10. 4. · tumor and stem...

2010;70:8874-8885. Published OnlineFirst October 19, 2010.Cancer Res Matilde Todaro, Flora Iovino, Vincenzo Eterno, et al. Stem CellsTumorigenic and Metastatic Activity of Human Thyroid Cancer

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e Todaro1, Flora Iovino1, Vincenzo Eterno1,4, Patrizia Cammareri1,4, Guido Gambara5,6,
ia Espina5, Gaspare Gulotta2, Francesco Dieli3, Silvia Giordano7, ro De Maria6,8, and Giorgio Stassi1,4
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roid carcinoma is the most common endocrine malignancy and the first cause of death among endo-ancers. We show that the tumorigenic capacity in thyroid cancer is confined in a small subpopulation ofike cells with high aldehyde dehydrogenase (ALDHhigh) activity and unlimited replication potential.high cells can be expanded indefinitely in vitro as tumor spheres, which retain the tumorigenic potentialdelivery in immunocompromised mice. Orthotopic injection of minute numbers of thyroid cancerells recapitulates the behavior of the parental tumor, including the aggressive metastatic features oferentiated thyroid carcinomas, which are sustained by constitutive activation of cMet and Akt in thyroidr stem cells. The identification of tumorigenic and metastagenic thyroid cancer cells may provide
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unprecedented preclinical tools for development and preclinical validation of novel targeted therapies.Cancer Res; 70(21); 8874–85. ©2010 AACR.

receptstreamGSK3βsignalmeseninvasithelialtors Tactivatumoractivaand trwhichcapacThe

duction

roid cancer is the most frequent endocrine malignancyglobal increasing incidence. Papillary (PTC), follicular

, and anaplastic (UTC) thyroid carcinomas arise fromermal-derived follicular cells, which represent the mostant cellular population of the thyroid gland. PTC com-80% to 85% of all thyroid neoplasms, whereas FTC iscond most common thyroid cancer, accounting forximately 10% to 15% of cases (1). The least common) histotype is UTC, which has a fast progression andpoor prognosis (2, 3).osine kinase receptors play a major role in the regula-f tumor initiation and progression (4, 5). In response toocyte growth factor/scatter factor, the Met tyrosine

triggers intracellular signals that positivelyvival differentiation and invasion (6, 7). Met

of tumpeuticcharaca crucUnt

origincells athrougtenceassumdiffereand pgenesthe rethyroiformacells,UTC,

s: 1Department of Surgical and Oncological Sciences,cular Pathophysiology Laboratory, 2Department ofiopathology and Biomedical Methodologies, Universityo, Italy; 4Cellular and Molecular Oncology, IRCCSre Maugeri, Pavia, Italy; 5Center for Applied Proteomicsicine, George Mason University, Manassas, Virginia;matology and Oncology, Istituto Superiore di Sanità,ute for Cancer Research and Treatment, University ofool, Candiolo, Turin, Italy; and 8Mediterranean Instituteia, Italy

ry data for this article are available at Cancer Researchrres.aacrjournals.org/).

uthors: Giorgio Stassi or Ruggero DeMaria, Phone:39-0382-592065; Fax: 39-091-6553238/[email protected], [email protected], or [email protected].

5472.CAN-10-1994

ssociation for Cancer Research.

21) November 1, 2010

Research. on May 16, 2013cancerres.aacrjournals.org aded from

or activates phosphatidyl inositol-3-kinase and down-kinases critically involved in cell survival, such asand Akt (8–10). Phosphatidyl inositol-3-kinase/AKT

transduction pathway is also involved in the epithelial-chymal transition, a process that confers motility andveness to epithelial tumor cells through the loss of epi-proteins and the upregulation of the transcription fac-wist and Snail (11). Strong evidence depicts aberranttion of β-catenin and Met in a wide variety of humans, including thyroid cancer (12, 13). Overexpression ofted β-catenin plays critical roles in both cell adhesionanscriptional regulation in the Wnt signaling pathway,has been implicated in the maintenance of self-renewality of the stem cell compartment (14).recent discovery of cancer stem cells (CSC) in a varietyors has changed the view of carcinogenesis and thera-strategies (15, 16). Therefore, the identification andterization of such tumorigenic population representsial step to develop effective therapies.il a few years ago, thyroid carcinoma was believed toate from well-differentiated normal thyroid folliculars a consequence of multiple mutations accumulatedhout the entire life span (17). More recently, the exis-of several degrees of differentiation has lead to theption that a pool of stem cells at different stages ofntiation are responsible for thyroid cancer initiationrogression. A novel hypothesis of thyroid carcino-is posits that thyroid cancer cells are derived frommnants of fetal cells (18). According to such a theory,d cancer cells would be generated from the trans-tion of three types of fetal thyroid cells—thyroid stem
thyroblasts, and prothyrocytes, which would result inPTC and FTC, respectively. Although the phenotype
. © 2010 American Association for Cancer


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and/or progenitor cells of the thyroid gland has noten identified, several markers including CD133 (19–23),(24–27), and ALDH1 (28–32) have been associated witherentiated cells in several tissues. Increased aldehyderogenase (ALDH) activity has been described in primi-lls from multiple myeloma (30), acute myeloid leukemiaancreatic (29), breast (28), and lung (32) carcinomas.e, we analyzed the tumorigenic activity of thyroid can-lls. We found that FTC, PTC, and UTC contain a smallation of tumorigenic cells that can be prospectivelyied through the ALDH activity. Thyroid cells with highexpression (ALDHhigh) possess the ability to self-renewinitiate serial transplantable tumors that recapitulateenotype and metastatic behavior of parental tumorsted by the activation of Met and Akt.

rials and Methods

s, cell culture, clonogenic and invasion assaysroid cancer tissues were obtained at the time of thy-tomy from patients affected by PTC (n = 18; age range,y), FTC (n = 10; age range, 21–72 y), and UTC (n = 6;nge, 51–73 y). Normal and tumor thyroid cells were pu-from fresh tissues as described in ref. (33). To obtaind spheres, cells were resuspended in medium contain-sic fibroblast growth factor and epidermal growth(20, 22) and plated on ultra low-adhesion 96-well platesoncentration of a single cell per well. Wells containingnone or more than one cell were excluded for the anal-or sphere-derived adherent culture, tumor spheres wereed in adherence conditions in DMEM supplemented0% fetal bovine serum or with human recombinantoid-stimulating hormone (400 ng/mL; Genzyme).migration was measured using growth factor–depletedel-coated (BD Biosciences) transwell inserts. Dissociatedcells (1.5 × 103) were plated onto Matrigel-coated trans-ith 8 μm pore size. DMEM supplemented with 5% ofserum was plated in the lower compartment of theell. After plating, migrated cells were counted up tors.

nohistochemistry and immunofluorescenceimmunohistochemical analysis, slides were heated forn retrieval in 10 mmol/L of sodium citrate (pH 6.0).ns were subsequently exposed to specific antibodiesroglobulin (Tg, DAK-Tg6; Dako), TTF1 (SPT24; Novo-), cytokeratin 19 (CK19, RCK108; Dako), CD133/13; Miltenyi), CD44 (DF1485; Biogenex), ALDH11, 44; BD Biosciences), or isotype-matched controlsropriate dilutions. Then, sections were incubated withylated immunoglobulins and treated with streptavidin-dase following the instructions of the manufacturer2 Kit; Dako). Stainings were revealed using 3-amino-lcarbazole substrate and counterstained with aqueousoxylin.immunofluorescence, cytospins of thyroid spheres or
-derived adherent cells were fixed with 2% paraformal-e, permeabilized with 0.1% Triton X-100, and exposed
obtainthe in

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primary antibodies. Cells were then labeled with FITC-damine red–conjugated secondary antibodies (Invitro-lus RNase (200 μg/mL; Sigma). Counterstaining wasmed using Toto3 iodide (642/660; Invitrogen).

cytometry and ALDEFLUOR assayshly isolated thyroid cells were permeabilized with thex/Cytoperm Kit (BD), exposed to antibody primarydies or corresponding isotype controls, rinsed, andd with secondary antibodies. Analysis was performeda flow cytometer.ification of ALDHhigh cells was performed using theFLUOR kit (StemCell Technologies). As a negativel, an aliquot of cells from each sample was treated0 mmol/L of diethylaminobenzaldehyde, a specificinhibitor. Intracellular fluorescent product was mea-by flow cytometry and sorting gates were establishedthe negative controls.

titative and reverse transcription-PCR analysisal RNA was obtained using the RNeasy Mini kit (Qiagen) and reverse transcribed using the High-Capacity cDNAe kit (Applied Biosystems). Quantitative TaqMan PCRis was performed with the ABI PRISM 7900HT Sequenceion System (Applied Biosystems) in a reaction volumeL containing 1× TaqMan Universal Master Mix (Appliedtems) and 1× probes and primer sets Hs0016745_m11a1, TaqMan Gene Expression Assays; Applied Bio-s) or 1× human glyceraldehyde-3-phosphate dehydro-e (Pre-Developed TaqMan Assay Reagents; Appliedtems). Data processing and statistical analysis weremed using the ABI PRISM SDS, software version 2.1ed Biosystems).

noblottingates were fractioned on SDS-polyacrylamide gels andd to nitrocellulose. Membranes were blocked for 1 houronfat dry milk and successively incubated with anti-specific for Met (sc-161, C20, rabbit polyclonal; Santa

Biotechnology), pMet (D26, Tyr1234/1235, rabbit IgG;AKT (9272, rabbit polyclonal; CST), pAkt (9271, Ser473,polyclonal; CST), E-cadherin (4065, rabbit polyclonal;and β-actin (Ab-1 mouse IgM; Calbiochem). Membraneshen washed, incubated for 1 hour with horseradish per-e–conjugated anti-mouse or anti-rabbit immunoglobu-mersham), and developed with a chemiluminescenceion system (Pierce Chemical, Co.).

ction of lentiviral particles and infectione transfer was performed using a TWEEN lentiviralcontaining luciferase (LUC) and green fluorescent pro-FP) as reporter genes (34). Transfection of packaging

n embryonic kidney cell line HEK-293T was assessedFuGENE 6 Reagent (Roche) and following the instruc-of the manufacturer.le short hairpin RNA (shRNA) expressing UTC cells were
ed by infection with pLK01 lentiviral plasmid containingterfering sequence of puromycin-resistant Akt (Sigma).
Cancer Res; 70(21) November 1, 2010 8875



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ells were selected with puromycin drug (2 μg/mL, Sigma)days to enrich for positive transfectants. ShMet (pCCLsin.PGK.GFP.Wpre) was obtained as described in ref. (33)

al model-week-old nude mice from Charles River Laboratoriesaintained in accordance with the institutional guide-f the University of Palermo animal care committee.r s.c. injection, tumor size was measured using thela: (π/6) × larger diameter × (smaller diameter)2. Foropic xenografts, 6-week-old nonobese diabetic/severeined immunodeficiency mice were injected into thethyroid gland using a 25 μL Hamilton syringe and age needle with the support of a dissecting microscope.ce were analyzed on a weekly basis by in vivo imagingace Lab) upon i.p. injection (100 μL) of D-lucifering/mL; Sigma-Aldrich; ref. 35).

tical analysisawere expressed asmean± SD. Statistical significancewasined by ANOVA (one-way or two-way) with Bonferroniest. Results were considered significant when P valuesess than 0.05 (*, P < 0.05; **, P < 0.01; and ***, P < 0.001).

lts

ll population of thyroid carcinoma cells retainsgenic capacitymeasured the clonogenic activity of tumor cells freshlyed from thyroid tumors. Limiting dilution analysisd that the mean number of clonogenic cells was 2% in.2% in FTC, and 3.5% inUTC. This small cell subset couldanded unlimitedly as thyrospheres after serial passages,as the remaining cells displayed a limited growth thatt last more than 2 weeks (Fig. 1A and B; data not shown).gh we found some CD133 transcripts in normal thyroidTC cells, clonogenic thyrosphere cultures expressed1 and CD44 at the protein level, whereas CD133 andwere essentially negative in all the cases examinedC; Supplementary Fig. S1A–B). In accordance with itserentiated phenotype, UTC sphere cultures did nots Tg, whereas PTC and FTC spheres showed a diffusesion (Fig. 1C; Supplementary Fig. S1B). In the presenceum or thyroid-stimulating hormone, the free-floatingpheres adhered and gradually acquired the expressionkeratin 19 (CK19; Fig. 1D; Supplementary Fig. S1B–C),increasing the content of Tg and maintaining an equalsion of CD44 (Fig. 1E). In contrast, the expression of1 was almost abolished after 5 days of culture in PTCC adherent cells, whereas UTC cells required a longerre to serum to reduce ALDH1 expression (Fig. 1E and F;mentary Fig. S1D). Despite acquiring a newmorphologyadherent conditions (Supplementary Fig. S1D), UTCs could be re-derived from sphere-derived adherentp to 10 days of culture in the presence of serum, follow-eeding in nonadherent conditions (data not shown).
unofluorescence analysis revealed that the stem cellOct3/4 and Nanog were expressed in all three histo-
phenojected

r Res; 70(21) November 1, 2010


ariants of thyroid carcinoma sphere cells, dramaticallyed in adherent cells from PTC and FTC, and main-in the majority of UTC spheres–derived adherent cellslementary Fig. S1E). Nestin was highly expressed innd FTC spheres and gradually lost in their respectives-derived adherent cells, whereas UTC cells were con-tly negative in both conditions.lectively, these data suggest that epithelial thyroid can-re hierarchically composed by a small cell populationlonogenic potential that gives rise to a more differen-progeny of the respective tumor.

expression is restricted to a small population ofid cells and its relative abundance correlatesalignancy

analyzed 18 PTC, 10 FTC, and 6 UTC (Table 1) for thece of putative cancer stem/progenitor cells. Immuno-hemistry and flow cytometry analyses of differentia-temness markers revealed distinct protein expressions among the different histologic thyroid cancer typesA and B). The terminal thyroid differentiation markerd the transcription factor TTF1 were detected in allC and FTC. In contrast, their expression was reducedsing in UTC, confirming the absence of thyroid-specificons in the latter (Fig. 2A and B). CK19 was preferentiallysed in PTC with respect to FTC, and was barely detect-cells purified from the UTC aggressive histologic vari-

ig. 2A and B). Analysis of the potential stem cell markersd that CD133 was not detectable (Fig. 2A), whereaswas constitutively expressed in all the thyroid tissues, both in tumors and their uninvolved pairs (Fig. 2A).trast, we observed the presence of rare ALDH1+ cellsmal tissues, which increased progressively in the moresive thyroid cancer histotypes (Fig. 2A). ALDH1+ cellsore abundant in UTC (16 ± 4%, n = 5) than in PTC (7, n = 12) and in FTC (3 ± 1.2%, n = 9; Fig. 2B–D). A smalltage (2%) of ALDH1+ cells was found in the UTC primaryent cultures (Supplementary Fig. S1B). Thus, ALDH1sion is restricted to a small population of cells of thed gland whose relative abundance in UTC may correlateigher malignancy.

with high ALDH expression are tumorigenic andduce the phenotypic characteristics of theal tumordetermine whether ALDH expression could be used aspective marker of thyroid tumorigenic cells, we isolat-DHhigh cells from all three histologic variants. As shown3A, cells with ALDHhigh expression were detected in allmors examined, with the highest percentage in freshlyiated UTC tissues (on average, 14 ± 3% of positiveThe analysis of cells from PTC and FTC revealed ar ALDHhigh fraction, accounting for 5 ± 2% and 2 ±f the whole-cell pool, respectively.rerequisite of putative CSCs is the ability to initiatedevelopment in recipient animals and reproduce the
type of the human parental tumor. Therefore, we in-ALDHhigh, ALDHlow, and unsorted cells isolated from
Cancer Research



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n thyroid cancers into the flanks of nude mice.gh not all the tumors contained cells able to initiated cancer in nude mice, ALDHhigh and unseparated cellsntly engrafted under the subcutis of the majority ofimals (ALDHhigh efficiency: 75% PTC, 70% FTC, andTC; Fig. 3B; Table 1). In all such animals, 5,000 ALDHhigh

ere more tumorigenic than 25,000 bulk cells, whereaslow

(in th

in PTC, FTC, and UTC spheres exposed for 5 d to fetal bovine serum (FBS). F,exposure to fetal bovine serum. Data are mean ± SD of five PTC, five FTC, and

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o form a tumor xenograft (Fig. 3B). The injection of anumber (25,000) of ALDHlow FTC and PTC cells didprove the tumor incidence. However, a limited andd tumor growth was observed 8 to 12 weeks fromlivery of 25,000 ALDHlow cells (Fig. 3B), as a possibleof the presence of contaminating ALDHhigh cells

e order of 0.2–0.3%) in the sorted ALDHlow fraction.low
LDH cells isolated from the same tumors were un- The failure of the ALDH population to generate tumors
1. A small population of thyroid cancer cells is clonogenic and forms spheres. A, percentage of clonogenic cells in freshly dissociated tumors.sentative phase contrast microscopy analysis of UTC single cell–derived spheres cultured in serum-free medium at the indicated days. C, confocalopy analysis of DIC (left) and immunofluorescence for the indicated antigens on thyroid cancer spheres. D, confocal microscopy analysis ofPTC, FTC, and UTC spheres–derived adherent cells cultured for 1 and 5 d. E, immunofluorescence analysis of thyroglobulin (Tg), CD44, and

ALDH1 mRNA expression in PTC, FTC, and UTC spheres beforethree UTC.




Table 1. Case description, tumorigenicity, and genetic alterations of thyroid cancer stem cells

Case Age/sex Tumor-node-metastasis Staging ALDHactivity (%)

ALDHHigh

spheresALDHHigh

xenograft formationBulk spheres

xenograft formationRET/PTC1 BRAF p53

PTC 1 44/F T2N0M0 I 4 Yes Yes Yes + − −PTC 2 28/F T2N0M0 I 3.5 No / Yes + − −PTC 3 41/F T2N0M0 I 3 No No / / / /PTC 4 30/M T4N1aM0 I 5 Yes Yes Yes − + −PTC 5 48/F T2N0M0 II 5.6 Yes Yes Yes − + −PTC 6 46/F T2N1aM0 III 4 No No / / / /PTC 7 70/F T1N0M0 I 4.5 No No / / / /PTC 8 31/F T2N0M0 I 3.9 Yes Yes Yes + − −PTC 9 30/F T2N0M0 I 4.3 Yes Yes Yes + − −PTC 10 36/F T3N0M0 I 4 No / / / / /PTC 11 57/F T2N0M0 II 5.2 Yes Yes Yes − + −PTC 12 39/F T2N0M1 II 5 Yes Yes Yes − + −PTC 13 56/F T2N0M0 II 4.5 Yes Yes Yes + − −PTC 14 73/F T4N0M0 III 4 Yes Yes Yes − + −PTC 15 32/F T2N0M0 I 5 Yes Yes / / / /PTC 16 39/M T2N0M0 I 6.2 Yes Yes Yes + − −PTC 17 28/F T2N0M0 I 4 No No / / / /PTC 18 62/F T1N0M0 I 4.2 Yes Yes Yes − − −FTC 1 52/F T2N0M0 II 2 Yes Yes Yes − − −FTC 2 71/F T2N0M0 II 1.8 No No / / / /FTC 3 68/F T4N1bM1 IV 3.2 Yes Yes Yes − − −FTC 4 21/F T2N0M0 I 2.2 Yes Yes Yes − − −FTC 5 53/F T2N0M0 II 2 Yes No Yes − − −FTC 6 32/F T2N1aM0 I 1.8 No No / / / /FTC 7 38/F T2N0M0 I 2 Yes Yes Yes − − −FTC 8 49/M T2N0M0 II 2.8 Yes Yes Yes − − −FTC 9 72/F T2N0M0 II 3 Yes Yes / / / /FTC 10 37/F T2N0M0 I 2 Yes Yes Yes − − −UTC 1 68/F T3N1aM1 IV 14 Yes Yes Yes − + +UTC 2 57/F T2N1bM1 IV 12.5 Yes Yes Yes − − +UTC 3 55/F T2N1bM1 IV 10.8 No No / / / /UTC 4 71/F T2N1aM1 IV 15.2 Yes Yes Yes − − +UTC 5 51/F T2N1aM1 IV 13 Yes Yes Yes − − +UTC 6 63/F T3N1aM1 IV 16 Yes Yes Yes − + +

NOTE: The symbol (/) indicates that the experiments was not performed.

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Figure(3-aminhematocells evrepresentative cytofluorimetric analysis for CK19 and ALDH1 performed on PTC, FTC, and UTC cells. D, mRNA expression of ALDH1 in tissues fromcontrol UTC.


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ing xenotransplantation was not due to a total depletioncer cells during the cell sorting procedure because thismorigenic cell population was mutated for BRAF in 512 PTC and in 2 out of 6 UTC (Supplementary Fig. S2A;1; data not shown). Interestingly, the resultant tumorrafts reproduced the genotypic and phenotypic charac-cs of their respective parental tumors, as shown bygic and immunohistochemical analyses (Fig. 3C; Sup-ntary Table S1). The presence of BRAF mutation wasated with a lower ALDH activity than RET/PTC1 rear-ent and p53 mutations, whereas the combination ofand p53 mutations was associated with the highestactivity in the xenografts (Supplementary Table S1).sistently, the highly tumorigenic ALDHhigh UTC cellsted CK19 and Tg-negative undifferentiated tumors,ducing the same phenotypic characteristics of the

, PTC, FTC, or UTC. Data are mean ± SD of five PTC, five FTC, and three

al tumor (Fig. 3C1). PTC, FTC, and UTC primary xeno-retained a similar amount of ALDH1 expression of the

(Fig. 4gener

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tal tumors (Fig. 3C; Supplementary Fig. S3A). Thus, aLDH expression characterizes the tumorigenic thyroidr population.

high thyroid cancer spheres retainr-initiating capacityfirst evaluated the self-renewal capacity of the total,high, and ALDHlow thyroid cancer cell populations byg freshly isolated tumor cells at clonal density underfferentiating conditions. In such culture conditions,high cells generated viable spheres and showed signifi-phere enrichment as compared with an equal numberseparated cells (Fig. 4A). Although at 21 days, 2 ×DHhigh cells from PTC, FTC, and UTC primary tumorsd 490 ± 100, 330 ± 74, and 550 ± 80 thyrospheres, re-vely, very few of the ALDHlow cells generated spheres

2. A small ALDH1+ population is present in thyroid carcinomas. A, H&E staining and immunohistochemical analysis for the indicated antigenso-9-ethylcarbazole, in red) on paraffin-embedded sections of human control thyroid gland, PTC, FTC, and UTC. Nuclei were revealed byxylin. One representative of 20 different controls, 18 PTC, 10 FTC, and 5 UTC are shown. B, percentage of Tg, CK19, TTF1, and ALDH1-positivealuated by flow cytometry. Data are mean ± SD of 15 independent experiments for control, 12 for PTC, 9 for FTC, and 5 for UTC. C, one

A; Supplementary Fig. S2B). The analysis of the clonesated from cells undergoing flow cytometry sorting




indicaclonogthe cloof clothe orspectispherrenewwere Amentaretainas thymice ktions20 dayUTC c

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Todaro et al.

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ted that the sorting procedure reduced the number ofenic cells from PTC and FTC by 50% without affectingnogenic performance of UTC cells. Thus, the numbernogenic cells within the ALDHhigh population was inder of 40%, 60%, and 25% for PTC, FTC, and UTC, re-vely. Clonogenic analysis showed that thyroid cancere cells display a considerably higher degree of self-al than normal thyroid sphere cells (Fig. 4B), whichLDH1+ and CK19− as the tumor counterparts (Supple-ry Fig. S2C). To determine whether ALDHhigh cellsed their tumorigenic potential after growing in vitrorospheres, we injected ALDHhigh spheres into nudeept for three passages under nondifferentiating condi-or grown in monolayer under adherent conditions for
s. PTC and FTC adherent cells did not engraft whereas roid c
ent of four performed with cells from different donors is shown. c1, representatiUTC ALDHHigh cells. No tumor growth was found at the UTC ALDHLow cells' (5



the small number of ALDHhigh cells that persistedadherent conditions (Fig. 4C; data not shown). In con-all thyroid cancer spheres were highly tumorigenic, ass invariably formed following the injection of as few as3 cells (Fig. 4C).eated in vitro passages of thyrospheres for up to 1 yeart affect cell growth or the ability to generate tumors innocompromised mice (Supplementary Fig. S2D). Fur-ore, serial transplantation experiments were conductedrepurified ALDHhigh spheres expanded in vitro from pri-secondary, and tertiary PTC, FTC, and UTC xenograftslementary Fig. S2E and F). Analysis of the clonogenicy and ALDH1 expression indicated that the number ofigenic cells did not change significantly in primary thy-
ancer xenografts as compared with the corresponding
ells formed very small tumors, which could be derived parental tumors (Fig. 2A; Supplementary Fig. S2G), suggesting

3. ALDHhigh cells retain tumorigenic capacity. A, representative FACS analysis of 7 PTC, 7 FTC, and 4 UTC cells using ALDEFLUOR assay.cancer cells exposed to ALDEFLUOR substrate (BAAA) and specific inhibitor of ALDH (DEAB) were used to define the population with highctivity (R2, green region) and population with low ALDH activity (R1, red region). B, size of tumors developed following s.c. injection of differentcancer subpopulations. Data are mean tumor size ± SD of five tumors per group derived from four separated patients. C, histologic andhistochemical analysis of paraffin-embedded sections of xenografts obtained 12 wk after injection of 5 × 103 ALDHHigh cells. One representative

ve s.c. tumor growth in nude mice 12 wk after injection of× 103 cells) injection site.

Cancer Research



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hyroid sphere–derived xenografts maintain the samechy as thyroid cancer. However, the number of tumori-cells together with the tumor growth rate tended to in-with serial xenografting, as the possible result of aon process (Supplementary Fig. S2F and G and S3B).

high-thyroid cancer spheres form tumors andtasis when orthotopically injected into mouseid glanddevelopment of experimental models of thyroidable to reproduce the human condition would be aful tool for preclinical validation of new therapies,ularly for the most aggressive histotypes. We injectedlls from PTC, FTC, and UTC spheres into the thyroidof immunocompromised mice. This procedure resulted% tumor formation efficiency within a few weeks fromon. In vivo imaging and microscopic examinations ofd tumors generated after 4 weeks by ALDHhigh PTCs revealed local tumor growth, whereas FTC spheresined thyroid gland infiltration and compression ofnt structures such as larynx and trachea (Fig. 5A). Sim-

eres-derived adherent cells (20 d of exposure to fetal bovine serum). Datat patients.

the injection of ALDHhigh UTC spheres resulted in tumorpment with a rapid invasive growth causing tracheal

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sophageal compression (Fig. 5A), thus recapitulatingnical features of this tumor.Mice injected with UTC cellse moribund and cachetic within 4 weeks after injectionthe impediment of the upper aerodigestive tract.

ogic examination of tumor specimens confirmed com-on and infiltration of mouse thyroid gland, trachea,ophagus (Fig. 5A and B). Tumorigenicity of orthotopi-njected thyroid cancer spheres was significantly fasterafter s.c. xenografting, even following serial in vivoes at the ectopic location (Supplementary Fig. S3B).reover, mice injected with ALDHhigh UTC spheresited cervical lymph node metastasis and a highf lung metastasis, whereas animals inoculated withhigh FTC spheres generated a moderate local invasiont regional or distant metastases (Fig. 5A and B). Immu-ochemistry and flow cytometry analyses of tumors andtases of mice orthotopically injected with ALDHhigh

pheres showed a significant enrichment of the putativeigenic ALDH1+ cells in lung metastases as comparedhe thyroid tumor (Fig. 5B; Supplementary Fig. S3A).r to parental tumors, Tg and CK19 differentiation

ean ± SD of 12 tumors per group generated with cells from three

4. Self-renewal of thyroid cancer stem cells. A, sphere-initiating capacity of freshly isolated cells with high (AF+) or low ALDH (AF−) activity, asred with unsorted (Bulk) cells. Data are mean ± SD of 10 independent experiments for PTC, 7 for FTC, and 4 for UTC with cells grown for 60 d.entage of sphere formation up to 60 d of normal (control) PTC, FTC, and UTC-dissociated sphere cells. C, tumorigenic potential of sphere cells or

rs were expressed in PTC and FTC orthotopic xeno-(Fig. 5B). These findings show that the orthotopic




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on of ALDHhigh thyroid cancer spheres reproduces theal and pathologic behavior, including tracheal andageal invasion, and high incidence of local and distanttases, which are peculiar characteristics of poorly dif-iated human thyroid tumors.

nd Akt activation drives tumorigenicity andtatic activity of thyroid cancer stem cells
define the metastatic potential of thyroid cancer stem Moreo


modifications of molecules commonly associatedthe metastatic signaling pathways (Supplementary4A). Reverse phase protein microarray technologymunoblot validation revealed a considerable upregu-of cMet, β-catenin, and E-cadherin, with an increasedhorylation status of cMet and Akt in sphere cells froms compared with those from normal thyroid (Control)nd FTC (Fig. 6A; Supplementary Fig. S4A and B).
ver, UTC sphere cells showed a higher percentage
(Supplementary Fig. S4C)

ures fon oy immer insoci

(bmoresegronds;H,unH1minaffinuseanmoane pmal mouse thyroid.

pulation, we quantitatively measured the posttransla- of β-catenin nuclear accumulation

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. © 2010 American Associati

5. Thyroid cancer sphererm tumors and metastasisrthotopic injection. A, wholeaging analysis of primarytastatic thyroid tumorsjection of 100 cells fromated PTC, FTC (top), andottom) spheres intouse thyroid gland. Onentative experiment of eightup is shown. SG, salivaryT, thyroid tumor, L, lung,heart. B, H&E staining andohistochemical analysis of, Tg, or CK19 revealed byo-9-ethylcarbazole (red) on-embedded sections ofthyroid gland invaded byPTC, FTC, and UTC cells,use lung metastasis byUTC cells. Experimentserformed as in A. Thy,

Cancer Research

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owed a higher migration capacity than the stem cellshe other thyroid tumor histotypes (Fig. 6B). Interest-migrated UTC stem cells showed pMet and pAKTulation, nuclear accumulation of β-catenin, andlete loss of E-cadherin expression (SupplementaryD). To understand whether Met and AKT activationlved in the regulation of CSC migratory ability, weregulated Met and AKT expression levels in UTCs. Transduction of spheres with lentiviral vectors en-specific Met and AKT shRNA sequences resulted insed amount of these two proteins and in efficientdown of their activated forms (Fig. 6C). We observed
unctional blockade of these molecules dramaticallyd the migration of UTC sphere cells (Fig. 6D). More-
in the(Fig. 6

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nockdown of MET and AKT significantly reduced thelevels of Snail and Twist (Supplementary Fig. S5A),

ey transcription factors that characterize epithelial-chymal transition.next determined whether Akt and Met silencing mightin vivo therapeutic benefits. UTC sphere cells trans-with ShMet or ShAkt were grown orthotopically intothyroid gland. Met knockdown delayed tumor out-

h by about 9 weeks whereas ShAkt dramaticallyed the capacity to give rise to orthotopic tumorsated by UTC stem cells (Fig. 6E, left; SupplementaryB). Moreover, silencing of either Met or Akt resulted

6. Met and Akt activity promotesenic and metastatic capacity of thyroidstem cells. A, immunoblot for pMet,kt, Akt, and E-cadherin in normal thyroidl), PTC, FTC, and UTC sphere cells.ber of migrated PTC, FTC, and UTCcells through Matrigel and a membranem pore size after 48 h. C, representativeblot analysis of Met, pMet, Akt, andUTC sphere cells transduced withshRNA (Scrambled), Met (shMet), orNA (shAkt). D, migration analysis asUTC sphere cells transduced withled, shMet, or shAkt. E, whole-bodyimaging analysis of orthotopic tumorand lung metastasis at the indicatedgenerated by UTC sphere cellsced with Scrambled, shMet, or shAkt.e representative of four independent

complete abrogation of the metastatic capacityE, right; Supplementary Fig. S5B). These results clearly




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te that Met and/or Akt could be very promisingeutic targets in the treatment of thyroid cancer

se they play a role both in CSC growth and invasion.

ssion

e, we showed that thyroid cancer contains a populationorigenic cells that can be considerably enriched basedexpression of ALDH. Our data show that ∼1.2% to 3.5%whole thyroid cancer population are ALDHhigh tumor-cells that can be clonally expanded in vitro in serum-edium. A considerable percentage of ALDHhigh cells cannew and grow unlimitedly as thyroid spheres able tote serial tumor xenografts in immunocompromisedDuring thyroid sphere formation, single clonogenichigh cells undergo symmetrical and asymmetricaln, giving rise to other clonogenic cells together with ay of cells with limited proliferation potential.n exposure to serum or thyroid-stimulating hormone,enic cells gradually lose the expression of ALDH, to-with the tumorigenic potential. In such differentiatingions, PTC and FTC ALDHhigh cells undergo aberrantntiation, as indicated by the acquisition of CK19 andregulation, whereas UTC cells remain largely undiffer-ed. Sphere-derived tumor xenografts reproduce theal tumor, both in terms of morphology and antigen ex-on. The pattern of ALDH1, CK19, and Tg expressione percentage of tumorigenic cells did not significantlyparental tumors and respective primary xenografts,

ting that thyroid cancer cells possess a functional hier-and heterogeneity.ious studies on malignant cancer development showede transcriptional program awarding migration and in-involves transcription factors belonging to Snail andfamilies (36), which play a similar role in the epithelial-chymal transition displayed in embryogenesis. Mets ligand are overexpressed in a variety of tumors.ctivation of Met induces proliferation, invasion, andenesis, contributing to tumor growth and spreading.roid cancer, Met activation has been proposed as ave prognostic factor (37). The current study revealsportant relationships between Met/Akt pathway andgressive phenotype of thyroid CSCs. Our data showMet and pAkt expression is greater in UTC than innd FTC. We found that activation of Akt, Met, andnin, together with downregulation of E-cadherin, con-otile and invasive behavior to UTC stem cells. Loss oferin at the invasive front was shown in a variety ofs during the transition from adenoma to carcinoma). Although targeting E-cadherin or its regulatory tran-on factors Snail and Twist would seem to be a goodgy, these factors are strictly essential for normal celly. Moreover, Snail has been shown to inhibit a greater of genes unrelated to E-cadherin downregulation,e-establishment of E-cadherin does not restore anlial phenotype (41). Interestingly, Akt or Met targeting
ses Twist and Snail expression, abolishing migrationetastatic activity of UTC stem cells. Our findings
ReceOnlineF



rt a role for Akt and Met targeting for the treatmentasive malignant thyroid cancers.cent study indicates that the evaluation of tumorigenictial in melanoma is strictly dependent on the systemor the assay (42). Several studies have relied on the useunocompromisedmouse recipients to quantify the per-e of tumorigenic cells present in the tumors, which wered as ∼0.0001% in melanoma or 0.057% in colon cancer). At least in the case of melanoma, the system used foralculation has considerably undervalued the percentageorigenic cells, which seems to be on the order of 27%n our analysis of the tumor-initiating cells in thyroidr, we evaluated the percentage of clonogenic cells tothe cell subset endowed with tumorigenic potential.gh our assay may have underestimated the percentageor-initiating cells present in the tumors, both in vitrovivo analyses indicated that tumorigenic activity is con-n the ALDHhigh cell population, which may contain 25%of the clonogenic cells able to generate tumor xeno-

in immunocompromised mice after in vitro expansion.absence of reliable experimental models has delayedvelopment of new therapies for thyroid tumors. Be-thyroid cancer is particularly resistant to conventionaltherapy, the therapeutic procedures have remainedially the same in the last 20 years and rely on surgeryed by radioactive iodine treatment (44–46). However,anced thyroid cancer, surgery is unable to eradicatemor. Although the more differentiated forms can beby radioactive iodine treatment (47), poorly differen-histotypes often do not express the iodide symporterre resistant to this therapy (48).possibility of prospectively isolating and growing tu-

enic clones from thyroid cancer has considerable impli-s. An extensive characterization of tumor-initiatingay allow the identification of new biomarkers for

ostic and therapeutic purposes. Moreover, these cellse screened for drug sensitivity or used for generatinganimal models of thyroid cancer. In this context, theopic delivery of thyroid cancer stem cells is able to re-late the aggressive behavior of undifferentiated thyroidomas, including local and distant metastases. Becausestic thyroid tumors are invariably lethal, this ortho-model could be exploited for experimental testingreclinical validation of new treatments.

osure of Potential Conflicts of Interest

otential conflicts of interest were disclosed.

Support

rammi di Ricerca Scientifica di Rilevante Interesse Nazionale 2007 prot.8NFY (G. Stassi), Programma Straordinario di Ricerca Oncologica. Stassi and R. DeMaria) and Associazione Italiana per la Ricerca sul(G. Stassi, R. DeMaria, and M. Todaro).costs of publication of this article were defrayed in part by the paymentcharges. This article must therefore be hereby marked advertisement innce with 18 U.S.C. Section 1734 solely to indicate this fact.

ived 06/03/2010; revised 08/06/2010; accepted 08/10/2010; publishedirst 10/19/2010.

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