induction of the stem-like cell regulator cd44 by rho ... · hirokazu ohata 1, tatsuya ishiguro ,...

Tumor and Stem Cell Biology

Induction of the Stem-like Cell Regulator CD44 by RhoKinase Inhibition Contributes to the Maintenanceof Colon Cancer–Initiating Cells

Hirokazu Ohata1, Tatsuya Ishiguro1, Yuki Aihara1, Ai Sato1, Hiroaki Sakai1, Shigeki Sekine3,Hirokazu Taniguchi3, Takayuki Akasu4, Shin Fujita4, Hitoshi Nakagama2, and Koji Okamoto1

AbstractThe difficulty in expanding cancer-initiating cells in vitro is one of major obstacles for their biochemical

characterization.We found that Rho kinase (ROCK) inhibitors aswell as blebbistatin, amyosin II inhibitor, greatlyfacilitated the establishment of spheroids from primary colon cancer. The spheroid cells expressed cancer stemcell markers, showed the ability to differentiate, and induced tumors in mice. The spheroids were composed ofcells that express various levels of CD44, whereas CD44high cells were associated with increased sphere-formingability, expression of the activating form of b-catenin, and elevated levels of glycolytic genes, CD44�/low cellsshowed increased levels of differentiationmarkers and apoptotic cells. The spheroid cells expressed variant formsof CD44 including v6, and the induction of the variants was associated with the activating phosphorylation of c-Met. As expected from the predicted hierarchy, CD44high cells differentiated into CD44�/low cells. Unexpectedly, afraction of CD44�/low cells generated CD44high cells, and the ROCK inhibitor or blebbistatin primed the transitionby inducingCD44 expression.Wepropose that the transition fromCD44�/low toCD44high state helps tomaintain aCD44high fraction and the tumorigenic diversity in colon cancer. Cancer Res; 72(19); 5101–10. �2012 AACR.

IntroductionThe emerging picture from recent discoveries revealed that,

in some types of tumors, only a small fraction of cancer cells iscapable of initiating cancer (1–3). These cancer-initiating cells,or cancer stem cells (CSC), as they are often defined because oftheir associated characteristics with stem cells, are one ofthe major foci of recent cancer research (1–3). Elucidation ofthe biologic and biochemical nature of CSCs will be importantto understand the mechanisms of cancer development and todevise new strategies for cancer therapy.It has been reported that CSCs are present in colorectal

cancer (4, 5). Colon CSCs were identified as cells that expressspecific surface markers, including CD133, CD44, CD166, andALDH1 (4–14).Several laboratories reported that CSCs from colon cancer

proliferate in vitro as spheroids (5, 12, 14–18). Remarkably, thespheres can bemaintained in conditions of exponential growth

for more than a year without losing the ability to generatetumors (5). Characterization of the spheroids revealed that afraction of the cells that express surfacemarkers such asCD133(15, 17) or ALDH (16) are attributed to their features as cancer-initiating cells and that extrinsic factors such as IL-4 (15) orWnt (12) mediate maintenance of CSC population.

Despite of the progress on the characterization of the colonspheroid cells, their stable culture in vitro can be maintainedonly from a fraction of primary cancers (12, 15), and it will beinstrumental to establish more efficient methods for spheroidcultivation to clarify the common biochemical nature of coloncancer–initiating cells.

In this article, we found that inhibitors of Rho-associatedprotein kinase (ROCK) or of actomyosin cytokinesis markedlyfacilitated the formation of spheroids from primary coloncancers, and revealed that the CD44high cells in the spheroidsshare common characteristics with CSCs. Unexpectedly, afraction of CD44�/low cells was capable of developing intoCD44high cells via the induction of CD44 expression by theROCK inhibitor. We will discuss the potential significance ofthe reversible transition between CD44high and CD44�/low cellsin light of the plasticity of CSCs and of devising a noveltherapeutic strategy against colon cancer.

Materials and MethodsPrimary human colon cancer specimens

All human colon cancer samples were resected frompatients with informed consent at the National Cancer CenterHospital (Chuo-ku, Tokyo, Japan), and all procedures wereconducted under the protocol approved by the ethics com-mittee of the National Cancer Center.

Authors' Affiliations: 1Division of Cancer Differentiation and 2Division ofCancer Development System, National Cancer Center Research Institute;Departments of 3Pathology andClinical Laboratories, and 4GastrointestinalOncology, National Cancer Center Hospital, Chuo-ku, Tokyo, Japan

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors:Koji Okamoto, Division of Cancer Differentiation,National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo104-0045, Japan. Phone: 81-3-3542-2511; Fax: 81-3-3542-2530; E-mail:[email protected]; and Hitoshi Nakagama, E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-11-3812

�2012 American Association for Cancer Research.

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Isolation of cancer cells and cell cultureTumor samples were minced and enzymatically dissociated

with 1 mg/mL collagenase D (Roche) and 1 mg/mL DNase I(Roche) for 1 hour at 37�C, and then sequentially filteredthrough 100 and 70 mm cell strainers (BD Falcon). After thelysis of red blood cells with Red Blood Cell Lysis Solution(Miltenyi Biotec), the filtered cells were grown in STEMPROhESC SFM (Invitrogen) supplemented with 8 ng/mL bFGF(Invitrogen), 20 mmol/L Y-27632 (Wako), and penicillin/strep-tomycin on ultra-low attachment culture dishes (Corning). Forserial passage, spheroid cells were dissociated into single cellswith Accutase (Invitrogen) once a week and incubated underthe culture conditions described earlier. For differentiationexperiments, spheroid cells were cultivated in the presence of10% FBS on standard tissue culture dishes (BD Falcon).

Flow cytometry analysisDissociated single spheroid cells were filtered, incubated

with 7-AAD (BD Pharmingen) for the exclusion of nonviablecells, and double-stained with a phycoerythrin (PE)-conjugat-ed monoclonal antibody against CD44 (G44-26; BD Pharmin-gen) and an allophycocyanin (APC)-conjugated monoclonalantibody against CD133 (AC133; Miltenyi Biotec). Isotype-matched mouse immunoglobulins were used as controls.Stained cells were then sorted using the FACS Aria II CellSorter (BD Biosciences) under the following conditions: nozzletip diameter (100 mm), pressure (20 psi), and threshold rate(�2,000 events/s). Subsequently, the sorted cells were analyzedusing FlowJo ver.7.6 software. Viability of the sorted cells wasexamined by Trypan-blue staining.

For analyses of cell-cycle profiles of cells with different levelsof CD44 expression, dissociated cells were double-stained withPE-conjugated anti-CD44 and APC-conjugated anti-CD133antibodies, fixed with 70% ethanol, and incubated with 0.1mg/mL RNase A (Qiagen) and 25 mg/mL propidium iodide(Sigma), to determine theDNAcontents of double-stained cellsfrom each fraction with different levels of CD44 expression.

In vitro assays for spheroid growth and formationAccutase-dissociated single cells or fluorescence-activated

cell sorting (FACS)-sorted cells were seeded at a density ofviable 1,000 cells per well on 96-well ultra-low attachmentplates (Corning). Cell growth was quantified by measuring theamounts of cellular ATP from a pool of spheroid forming andnonforming cells (CellTiter-Glo Luminescent Cell ViabilityAssay; Promega). Spheroid formation was evaluated by count-ing the number of formed spheres (>�20 mm in diameter). Forquantification of the inhibitory effects of CD44 inhibition onspheroid formation, 10 mg/mL anti-CD44 neutralizing antibo-dies (IM7; Biolegend and 2C5; R&D Systems) or the controlantibody were added at the beginning of spheroid formation.Single-cell dilution assays were conducted as described earlier(12).

Animal experimentsFor cell transplantation assays of spheroid cells, the spher-

oids were dissociated into single cells with Accutase,suspended in 50 mL medium containing 50% Matrigel (BD

Biosciences), and used for subcutaneous injection with a 27Gneedle into the flank of NOD/SCID mice (Central Institute forExperimental Animals, Tokyo, Japan). All mouse procedureswere approved by the Animal Care and Use Committees of theNational Cancer Center and conducted in accordance withInstitutional policies.

ResultsROCK inhibitors markedly improve sphere-formingefficiency from primary colon cancer cells

To isolate and expand cancer-initiating cells from primarycolon cancer, we dissected and cultivated primary cancer cellsunder spheroid culture conditions (5). In an attempt to estab-lish the optimum conditions for the maintenance and growthof cancer-initiating cells, we examined the effects on spheroidformation of chemicals that were reported to be effective inpromoting the growth of normal or cancer stem cells (data notshown). It was previously reported that ROCK inhibitor pro-motes the survival of embryonic stem cells (19–23). Afterextensive screening, Y-27632, a ROCK inhibitor, stood out asa chemical that greatly facilitated spheroid formation from acolon cancer specimen (Fig. 1A).

ROCKi-IV, another ROCK inhibitor, was also effective forspheroid formation (Fig. 1B). The presence of Y-27632 at 10 to20 mmol/L, a concentration sufficient for maintenance ofembryonic stem cells (21), caused maximum enhancement ofspheroid formation (Fig. 1C and D), and extended in vitrocultivation revealed that Y-27632 was required for sustainedgrowth of spheroids (Fig. 1E). There was a striking increase in afraction of cells with sub-G1 DNA content in the absence of Y-27632 (Fig. 1F) indicating that Y-27632 protects spheroid cellsfrom apoptotic cell death.

Because Y-27632 was highly effective in establishingspheroids from the aforementioned case (hereafter referredas #6), we tested the same conditions for cultivation fromother cases of primary colon cancers. In aggregate, wesuccessfully cultivated spheroids that could be maintainedin vitro for 1 month in 10 of 16 cases examined (Supple-mentary Table S1). Continued passage revealed that 5 of 10spheroids could be further expanded over a period of morethan 6 months (Supplementary Table S1). In all 5 cases, inwhich spheroid culture could be sustained, the withdrawalof Y-27632 resulted in marked reduction of spheroid forma-tion (Fig. 1A; Supplementary Fig. S1A–S1C). ROCKi-IV wasalso effective for spheroid formation (#9 and #20; Supple-mentary Fig. S1D and S1E). As in #6, Y-27632 was requiredfor continued growth of the spheroids (#20; SupplementaryFig. S1F), and for the protection of spheroid cells fromapoptotic cell death (#19 and #20; Supplementary Fig. S1Gand S1H). Taken together, the presence of ROCK inhibitormarkedly facilitates the proper maintenance and growth ofspheroids from primary colon cancer.

Spheroids formed in the presence of ROCK inhibitorexpress colon CSCmarkers, differentiate into epithelial-like cells, and are capable of forming tumors in mice

We next examined whether spheroid cells formed in thepresence of Y-27632 share characteristics for CSCs. We

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Cancer Res; 72(19) October 1, 2012 Cancer Research5102




determined whether spheroid cells were capable of (i)expressing specific markers, (ii) differentiation, and (iii)tumor formation in immunocompromised mice, based onthe proposed criteria for CSCs (3). First, spheroid cellsexpressed colon CSC–specific markers, CD44 and CD133(#6 and #20, Fig. 2B; Supplementary Fig. S2B). Second,spheroid cells were capable of differentiating into epitheli-al-like cells if grown under differentiating conditions, basedon their morphology (#6 and #20, Fig. 2A; Supplementary Fig.S2A), reduction of CD44 and CD133, and induction of adifferentiation marker, cytokeratin 20 (CK20; #6 and #20; Fig.2B and Supplementary Fig. S2B). Third, xenograft experi-ments using immunocompromised NOD/SCID mice showedthat the injection of spheroid cells (1 � 103 cells) could formtumors that were histologically indistinguishable from theoriginal primary tumor or from its mouse xenograft (#6, #19,and #20; Fig. 2C and Supplementary Fig. S2C and S2D). Thus,spheroids included cells that meet the major criteria forCSCs.

CD44high cells in spheroids show characteristics of CSCsTo gain insight into whether entire spheroid cells or only a

fraction retain characteristics associated with CSCs, we exam-ined the expression of CD44 and CD133 in spheroid cells byflow cytometry analysis. Although levels of CD133 expressiondid not significantly differ, there were striking differences inCD44 expression among cells (#6 and #20; Fig. 3A and Sup-plementary Fig. S3A). To determine whether the difference inCD44 expression reflects cellular hierarchy among them, wesorted spheroid cells into CD44�/low, CD44med, and CD44high

fractions. As expected, the sorted CD44high cells expressed highlevels of CD44, whereas their expression in CD44�/low was notdetectable by Western blot analyses (Fig. 3C). Analyses of thecell-cycle profile revealed that a significant fraction ofCD44�/low cells underwent apoptosis (#6 and #20; Fig. 3B andSupplementary Fig. S3B). CD44high cells could form spheroidsmore effectively than CD44�/low cells (Fig. 3D), and werecapable of differentiation based on the induction of CK20 anddownregulation of CD44 and CD133 (#6 and #20; Fig. 3E and

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Figure 1. ROCK inhibitors improve sphere-forming efficiency fromprimary colon cancer cells. A andB, bright-phase imagesof spheroids (#6) in thepresenceorabsence of 20 mmol/L Y-27632 (A) or 5 mmol/L ROCKi-IV (B). C, bright-phase images of spheroids (#6) in the presence of the indicated concentrationsof Y-27632. D, dose-dependent curves of the cell growth of spheroid cells (#6) by Y-27632 on day 3. Cell growth was quantified by measuring cellularATP. E, time course of spheroid cell growth (#6) in the presence or absence of 20 mmol/L Y-27632. F, suppression of cell death of spheroid cells (#6)by Y-27632. Spheroid cells were stained with propidium iodide, and a fraction of cells with sub-G1 DNA content was measured by flow cytometry analysis.

Regulation of CD44 by ROCK Inhibitor in Colon Cancer–Initiating Cells

www.aacrjournals.org Cancer Res; 72(19) October 1, 2012 5103




Supplementary Fig. S3C). In addition, CD44high cells gave rise toCD44med and CD44�/low cells under the conditions of spheroidculture (#6 and #20; Fig. 3F and Supplementary Fig. S3D),indicating that there is dynamic transition from CD44high cellsto CD44�/low cells in spheroids.

It was shown that high Wnt activity is associated with thecolon CSC phenotype (12). To examine whether high levels ofCD44 are associated with the activation of the Wnt pathway,we examined the activating phosphorylation of b-catenin(ser552; refs. 24–26) in the CD44high and CD44�/low cells. Thefraction of cells with the activating phosphorylation of b-cate-nin was higher in the CD44high cells than in the CD44�/low cells(Fig. 3G), which was corroborated by Western blot analyses(Fig. 3H). Thus, b-catenin was activated in the CD44high cells.

Enhancement of aerobic glycolysis is another phenotypichallmark associated with normal and CSCs (27–29). We con-ducted microarray gene expression analyses of the CD44high

and CD44�/low cells, and gene set enrichment analyses (30) ofthe expression profiles revealed that several pathways associ-ated with glycolysis, that is glucose metabolism and thepentose phosphate pathway, were upregulated (Supplemen-tary Fig. S3E). These are in agreement with recent reports (31),and indicate that the glycolytic metabolism is enhanced in theCD44high cells. Collectively, these results strongly suggest thatcells that express high levels of CD44 are associated with theknown CSC-like characteristics.

Inhibition of CD44 suppresses spheroid formationTo show that high levels of CD44 expression are required for

the CSC-like properties, we inhibited CD44 with the corre-

sponding shRNAs and determined whether inhibition of CD44thwarts spheroid formation. Knockdown of CD44 by theshRNAs was confirmed by quantitative reverse transcription(qRT)-PCR (#6 and #20; Fig. 4A and Supplementary Fig. S4A)and Western blot analyses (#6 and #20; Fig. 4B and Supple-mentary Fig. S4B). In accordance with the elevated levels ofapoptotic cell death in CD44�/low cells (Fig. 3B), the inhibitionof CD44 by shRNAs induced apoptotic cell death (data notshown). Examination of a remaining infectant indicated thatthe inhibition of CD44 by shRNAs reduced spheroid formation(#6 and #20; Fig. 4C and Supplementary Fig. S4C) as well as cellgrowth (Fig. 4D and Supplementary Fig. S4D).

In agreement with the inhibition of spheroid formation bythe CD44 shRNAs, spheroid formation and cell growth werecompromised by the inhibition of CD44 by neutralizing anti-bodies (#6 and #20; Fig. 4E and F and Supplementary Fig. S4Eand S4F). The results from serial dilution assays confirmed thatthe anti-CD44 antibody inhibited the clonal growth of #20spheroid cells (Supplementary Fig. S4G). Combined with thedata presented in Fig. 3, these data indicate that CD44 expres-sion is required to maintain the CSC-like characteristics ofCD44high cells.

ROCK inhibitor induces variant forms of CD44The functional importance of CD44 presented in Fig. 4

prompted us to examine its expression in spheroids. Of the5 established spheroids examined, 2 (#6 and #20) expressedhigher levels of CD44 than the others (#9, #17, and #19) in thepresence of Y-27632 (Fig. 5A). Strikingly, CD44 expression wasmarkedly reduced in the absence of Y-27632 in all the

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Figure 2. Spheroids formed in thepresence of ROCK inhibitorexpress colon CSC markers,differentiate into epithelial-likecells, and are capable of formingtumors in mice. A, bright-phaseimagesof spheroids (#6) before andafter differentiation (day 7). B,Western blot analyses of spheroidcells (#6) before and afterdifferentiation with the indicatedantibodies. Asterisk indicatesnonspecific band. C, hematoxylinand eosin staining of the primaryhuman colon tumor (left),subcutaneous xenograft of thesame primary tumor (center), andsubcutaneous xenograft of thespheroid cells (right). Spheroid cellswere derived from an identicalprimary tumor (#6). Scale bars,100 mm.

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spheroids, whereas CD133 expression was not significantlyaffected (Fig. 5A). The withdrawal of Y-27632 caused rapidreduction of CD44 within 3 days (#6 and #20; Fig. 5B andSupplementary Fig. S5A).In addition to the standard form (CD44s), there are several

variant isoforms of CD44 (CD44v) as a result of alternativesplicing (32). The molecular weight of the major polypeptidedetected with the anti-pan-CD44 antibody (�170 kD) indi-cated that CD44v was a major form induced by Y-27632 (#6and #20; Supplementary Fig. S5B and S5C, left columns).Western blot analyses with variant-specific antibodiesrevealed that CD44v9 and CD44v6 were induced by Y-27632 (Supplementary Fig. S5B and S5C, middle and rightcolumns). The molecular weight of the major band detected

with the anti-pan-CD44 antibody approximately matchedthat of CD44v9 (Supplementary Fig. S5B and S5C), althougha longer exposure revealed that the anti-pan-CD44 antibodydetected the polypeptide that roughly corresponds to thesize of CD44v6 (data not shown). Thus, several variant formsof CD44 were induced by Y-27632.

To determine whether CD44 expression can be reinitiatedafter readdition of the ROCK inhibitor, dissociated spheroidcells were cultivated in the absence of Y-27632 for 3 days, andthen reincubated with the inhibitor. The readdition of Y-27632resumed CD44 expression, and 10 to 20 mmol/L Y-27632 wassufficient for maximum induction of CD44 (#6; Fig. 5C). Time-course analyses of CD44 induction by Y-27632 showedthat CD44 was induced for 3 days after incubation (#6 and

Figure 3. CD44high cells in spheroidsshowcharacteristics of CSCs. A, flowcytometry analyses of spheroid cells(#6) double-stained with anti-CD44and anti-CD133 antibodies. B, thecell-cycle profiles of CD44�/low,CD44med, and CD44high cells. C,Western blot analyses of FACS-sorted CD44high and CD44�/low cellswith the indicated antibodies. D,bright-phase images of spheroids(day 3) of FACS-sorted CD44high andCD44�/low cells (left). Quantificationof spheroid formation (right). E,Western blot analyses of FACS-sorted CD44high cells (#6) with theindicated antibodies. CD44high cellswere harvested immediately (day 0)or cultivated for a week (day 7)under differentiation conditions.F, flow cytometry analyses of sortedCD44high cells (#6) cultivated inspheroid conditions for the indicatedperiods. G, immunofluorescencestaining of dissociated spheroidcells. Left, immunostaining with theindicated antibody. Right, a fractionof the spheroid cells that werepositive for staining with the anti-ser552 phosphorylated b-cateninantibody. H, Western blotanalyses of FACS-sorted CD44high

and CD44�/low cells with theindicated antibodies.

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#20; Fig. 5D and Supplementary Fig. S5D). ROCKi-IV as well asY-27632 was capable of inducing CD44 (#6 and #20; Supple-mentary Fig. S5E). The kinetics of the induction of CD44 wereoverall in line with those of spheroid formation (Fig. 1E anddata not shown). Thus, given the functional role of CD44expression in spheroid formation, these data strongly suggestthat the induction of CD44v contributes to the formation ofspheroids by ROCK inhibitors.

ROCK inhibitor primes the retrograde transition fromCD44�/low to CD44high cells

In accordance with the induction of CD44 after the read-dition of ROCK inhibitor, the incubation of FACS-sortedCD44�/low cells with Y-27632 caused an increase of CD44med

and CD44high cells (#6 and #20; Fig. 5E and SupplementaryFig. S5F). The induction of CD44-positive cells by Y-27632 wassupported by Western blot analyses (#6 and #20; Fig. 5F andSupplementary Fig. S5G). These results suggest that the ROCKinhibitor, by inducing CD44, induced reverse transition fromCD44�/low to CD44high cells in spheroids.

qRT-PCR analyses indicated that there was no significantincrease of CD44 mRNA over the same time course, indicatingthat CD44 was posttranscriptionally induced (#6 and #20; Fig.5G and Supplementary Fig. S5H).

Blebbistatin induces CD44 and promotes spheroidgrowth

It was reported that the enhanced survival of embryonicstem cells by ROCK inhibitors is mediated via the interfer-ence of the cytokinesis pathway, and that survival ofdissociated embryonic stem cells was greatly enhanced byBlebbistatin, an inhibitor of myosin hyperactivation, as wellas by ROCK inhibitor (20). Treatment of spheroid cells withblebbistatin, as well as with Y-27632, induced CD44 expres-sion (Fig. 5H and Supplementary Fig. S5I) and promoted cellgrowth of spheroid cells (Fig. 5I and Supplementary Fig.S5H). Thus, it is likely that the ROCK/myosin pathwaymediates the CD44-dependent enhancement of spheroidcell growth as well as cell survival of dissociated embryonicstem cells.

Expression of CD44 variants is associated with theactivating phosphorylation of c-Met

Todetermine the type of CD44 variant isoforms expressed inspheroid cells (Supplementary Fig. S5B and S5C), we con-ducted RT-PCR analyses using isoform-specific primers (Fig.6A; ref. 33). Overall profiles of CD44 isoforms were similarbetween those from the spheroids and HT29 cells (Fig. 6B andC), which were reported to express a variety of CD44 variantsincluding CD44v6 and CD44v9. Of note, CD44v6 mediates theactivation of the hepatocyte growth factor (HGF)/c-Met sig-naling (34). Indeed, CD44 induction by Y-27632 was associatedwith activating phosphorylation of c-Met (Fig. 6D), suggestingthat the induction of CD44v6 augments the HGF/c-Metsignaling.

CD44�/low cells are capable of forming CD44-positivetumors in mice

Finally, we examined the capacity of CD44high andCD44�/low cells to generate tumors in immunocompromisedmice. Both types of cells were capable of forming tumors,although the frequency of tumor formation by CD44�/low

cells was less than that by CD44high cells (SupplementaryFig. S6A). Hematoxylin and eosin staining showed thattumors derived from CD44high cells were histologically indis-tinguishable from the original primary tumor (#6; Figs. 2Cand 7A). CD44�/low cells also formed similar adenocarcino-ma, although cribriform and more PAS-positive vacuoleswere found in some parts of the tumor (Fig. 7A and data notshown).

Remarkably, some adenocarcinoma cells from CD44�/low

cells aswell as fromCD44high cells were CD44-positive (Fig. 7B),

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Figure 4. Inhibition of CD44 suppresses spheroid formation. A, qRT-PCRanalyses of CD44 expression in spheroid cells (#6) after infection withlentiviruses expressing the indicated CD44 shRNAs. B, Western blotanalyses of CD44 expression in the spheroid cells presented in A. C,spheroid formation after shRNA-mediated inhibition of CD44. Extent ofspheroid formation was measured by counting the number of spheroids.D, cell growth of spheroid cells after shRNA-mediated inhibition of CD44.Cell growth was quantified by measuring cellular ATP. E, spheroidformation after treatment with the indicated antibodies. F, cell growth ofspheroid cells after treatment with the indicated antibodies. C and D,1 � 103 cells were plated per well for 3 days, and average values from3 independent experiments are shown. �, P < 0.05; ��, P < 0.01;��, P < 0.001.

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suggesting that CD44�/low cells generated CD44-positivetumors. Supporting this, flow cytometry analyses showedthat a fraction of the CD44�/low cell-derived tumors as wellas CD44high cell-derived ones expressed CD44 (Fig. 7C).Comparison of CD44 expression between CD44�/low cell-derived tumor cells and the original FACS-sorted cellsindicated that approximately 10% of the CD44�/low cell-derived tumor cells became CD44-positive (SupplementaryFig. S6B). It is unlikely that CD44 expression in tumors fromCD44�/low cells was attributed to cells other than tumors,because CD44 staining was almost exclusively detected inthe tubular structure of adenocarcinoma (Fig. 7B). Westernblot analyses further confirmed the expression of CD44 intumors derived from CD44�/low cells (Fig. 7D). Judgingfrom levels of human Topo I expression, levels of CD44

expression in CD44high cell-derived tumors were approxi-mately 3-fold higher than those in CD44�/low cell-derivedones (Fig. 7D).

We also conducted transplantation assays using spheroidcells thatwere cultivated either in the presence or absence or Y-27632 (Fig. 5). The generated tumor from these cells showedsimilar CD44 staining (Supplementary Fig. S6C). Taken togeth-er, our data indicate that CD44-negative cells are capable offorming CD44-positive tumor.

DiscussionIn this report, we showed that the addition of ROCK inhi-

bitors markedly facilitated the continuous growth of cells withproperties for colon CSCs in vitro. Characterization of thecultivated cells showed that ROCK inhibitors were capable of

Figure 5. ROCK inhibitor primesretrograde transition from CD44�/low

to CD44high cells. A, Western blotanalyses of 5 spheroid cells in thepresence or absence of 20 mmol/LY-27632. The indicated spheroidswere grown under standard spheroidconditions or in the absence ofY-27632 for 7 days. Western blotanalyses were conducted with theindicated antibodies. B, Western blotanalyses of spheroid cells (#6) afterwithdrawal of Y-27632 for theindicated periods. C and D,dissociated spheroid cells (#6) wereincubated in the absence of Y-27632for 3 days and recultivated with theindicated concentration of Y-27632for 7 days (C) or with 20 mmol/L Y-27632 for the indicated periods (D).Western blot analyses wereconducted as described in A. E, flowcytometry analyses of FACS-sortedCD44�/low cells (#6) that were grownunder the spheroid conditions for theindicated periods. F, Western blotanalyses of sorted CD44�/low cells(#6) harvested immediately (day 0) orgrown under the spheroid conditionsfor 8 days. G, qRT-PCR analyses ofCD44 and CD133 of the spheroidcells (#6) described in D. GAPDHexpression was used as a control. H,Western blot analyses of spheroidcells incubated with the indicatedconcentration of Y-27632 orblebbistatin for 3 days. I, cell growthof spheroid cells (#6) grown underconditions described in H. Cellgrowth was quantified by measuringcellular ATP.

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sustaining spheroids that contained cells that met the majorcriteria for CSCs. In the formed spheroids, CD44high cellsshared characteristics for CSCs, and were capable of generat-ing their differentiated progeny, CD44�/low cells, as well asCD44high cells themselves.

Recently, it was reported that CD44v contributes tothe ROS resistance of gastrointestinal cancer stem-like cells(35), and associates with enhanced aerobic glycolysis (31).CD44 expression in spheroid cells is associated with theenhanced glycolytic pathways (Supplementary Fig. S3E),which may be attributed to CD44v expressed in the pres-ence of ROCK inhibitor (Figs. 6A–C and Supplementary Fig.S5B and S5C).

Of particular interest among the CD44 variants is CD44v6,because it was reported that CD44v6 mediates the activationof HGF/c-Met pathway (34). Indeed, our data indicate thatCD44 expression is associated with c-Met activation (Fig.6D). Considering that HGF restores the colon CSC pheno-type (12), CSC-like characteristics associated with CD44expression may be induced via the activation of the HGF/c-Met pathway.

What is the mechanism for the induction of CD44 byROCK inhibitors? It was reported that the functions ofROCK center on the regulation of cytoskeletons via thephosphorylation of its targets (36). Considering that CD44induction by the ROCK inhibitor is posttranscriptional (Fig.5D and G and Supplementary Fig. S5D and S5I), alterationsin the cytoskeleton caused by ROCK inhibitor may lead tostabilization or enhanced translation of CD44v protein.

Notably, ROCK inhibitors facilitate the in vitro growth ofembryonic stem cells by inhibiting dissociation-induced apo-ptosis (21), and the inhibition of the cell death is mediated viathe blockage of ROCK/myosin hyperactivation (20). Similarly,the inhibition of dissociation-induced apoptosis may also beimportant for the establishment of colon cancer spheroids, andindeed the inhibitor was used to block cell death of dissociatedorganoids that are derived from colon adenocarcinoma (18).ROCK inhibitors and blebbistatin may contribute to CD44-mediated spheroid formation through the inhibition of acto-myosin hyperactivation.

ROCK inhibitor may also regulate other pathways indepen-dent of CD44 induction to facilitate the formation of spheroids.In the absence of Y-27632, some spheroids (#6 and #20) expressresidual amounts of CD44, the levels of which are roughlyequivalent to those from other spheroids in the presence of theinhibitor (#9, #17, and #19; ref. Fig. 5A). Because the capacity toform spheroids was lower in the former than the latter (Sup-plementary Fig. S1C), the CD44 pathway and other unknownpathways may synergize to facilitate the formation ofspheroids.

The retrograde transition from CD44�/low to CD44high cellsby ROCK inhibitor may lead to the formation of a dynamicequilibrium between these cells. Of note, similar dynamictransitions between CSC and non-CSC states were alsoobserved in melanoma cells (37, 38) and in breast cancer cells(39, 40). It was proposed that, through mathematical analyses,stochastic transition between cancer stem-like cells and non-stem-like cells forms a dynamic equilibrium in cancer (39), and

A

B

Primer

Exon

D

C Figure 6. Expression of CD44variants is associated with theactivating phosphorylation of c-Met in spheroid cells. A, schematicpresentation of the position ofprimers for exon-specific RT-PCRanalyses. The forward primer islocated at the constant exon 5(C13) or each variant exon (v2-v10)of CD44, and the reverse primer islocated at the constant exon 15. B,RT-PCR analyses ofCD44 variants.C13 and the exon 15 primer areused to detect the variants from thespheroid cells and HT29 cells. RT-PCR analyses were conducted asdescribed before (33). C, RT-PCRanalyses with isoform specificprimers. Expression of each CD44variants was detected with theprimers shown in A. HepG2 cells,which do not express CD44, wereused as negative controls (33). D,induction of the activatingphosphorylation of c-Met in thepresence of Y-27632. The spheroidcells in the presence or absence ofY-27632 for 3 days were used forWestern blot analyses with theindicated antibodies.

Ohata et al.





such equilibrium may be influenced by extracellular factors(12). Thus, the phenotypic plasticity of CSC-like cells weobserved may be a widespread feature of solid cancer cells.In devising a therapeutic strategy against colon cancer–

initiating cells, tumors with high activity for retrograde tran-sition may pose a major problem, because elimination of bothCD44high and CD44�/low cells may be required for effectivetherapy; CD44�/low cells, if not simultaneously neutralized, willrevert back to CD44high cells with cancer-initiating ability. Thecombination of killing cancer-initiating cells and blockingretrograde differentiation may be considered an effectivetherapy in the future.

Disclosure of Potential Conflicts of InterestY. Aihara is employed (other than primary affiliation; e.g., consulting) in

Sysmex Corporation as a Researcher. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: H. Ohata, H. Nakagama, K. OkamotoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H. Ohata, T. Ishiguro, Y. Aihara, A. Sato, H. Sakai, S.Sekine, T. Akasu, S. FujitaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H. Ohata, H. Nakagama, K. OkamotoWriting, review, and/or revision of themanuscript:H. Ohata, H. Nakagama,K. Okamoto

Administrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): H. Taniguchi, H. NakagamaStudy supervision: H. Nakagama, K. Okamoto

AcknowledgmentsThe authors thank the laboratory members for critical reading of the

manuscript, Ibuki Kobayashi, and Naoaki Uchiya for technical assistance,Yasuhide Yamada for clinical suggestion, and Hideyuki Saya for anti-CD44v9antibody. SCADS inhibitor kit, which includes Y-27632, was a gift from theScreening Committee of Anticancer Drugs supported by Grant-in-Aid forScientific Research on Priority Area "Cancer" from MEXT, Japan.

Grant SupportThis study was supported by a Grant-in-Aid for the Third-Term Compre-

hensive 10-Year Strategy for Cancer Control from the MHLW (K. Okamoto); theProgram for Promotion of Fundamental Studies in Health Sciences of theNational Institute of Biomedical Innovation (NiBio; K. Okamoto); Grant-in-Aidfor Scientific Research in Innovate Areas fromMEXT (K. Okamoto); Grant-in-Aidfor Scientific Research (C) from Japan Society for the Promotion of Science (JSPS;K. Okamoto); Grant-in-Aid for Cancer Research from Foundation for Promotionof Cancer Research (K. Okamoto); Research Resident Fellowship from theFoundation for Promotion of Cancer Research (FPCR, Japan; T. Ishiguro).Grant-in-Aid for Young Scientist (B) from JSPS (H. Ohata and T. Ishiguro).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received November 30, 2011; revised June 19, 2012; accepted July 14, 2012;published OnlineFirst September 20, 2012.

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2012;72:5101-5110. Published OnlineFirst September 20, 2012.Cancer Res Hirokazu Ohata, Tatsuya Ishiguro, Yuki Aihara, et al. Initiating Cells

−Inhibition Contributes to the Maintenance of Colon Cancer Induction of the Stem-like Cell Regulator CD44 by Rho Kinase

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