Establishment and Characterization of an ... - Cancer Researchovarian cancer, and the established spheroids showed various characteristics of CSCs. We investigated functional roles

Tumor and Stem Cell Biology

Establishment and Characterization of anIn Vitro Model of Ovarian Cancer Stem-like Cellswith an Enhanced Proliferative CapacityTatsuya Ishiguro1,2, Ai Sato2, Hirokazu Ohata2, Yoshinori Ikarashi3,Ryou-u Takahashi4, Takahiro Ochiya4, Masayuki Yoshida5, Hitoshi Tsuda5,Takashi Onda6, Tomoyasu Kato7, Takahiro Kasamatsu7, Takayuki Enomoto1,Kenichi Tanaka1, Hitoshi Nakagama8, and Koji Okamoto2

Abstract

The establishment of cancer stem-like cell (CSC) culture sys-temsmay be instrumental in devising strategies to fight refractorycancers. Inhibition of the Rho kinase ROCK has been shown tofavorably affect CSC spheroid cultures. In this study,we showhowROCK inhibition in human serous ovarian cancer (SOC) cells canhelp establish a CSC system, which illuminates cancer patho-physiology and its treatment in this setting. In the presence of aROCK kinase inhibitor, spheroid cultures of SOC cells expressedcharacteristic CSC markers including ALDH1A1, CD133, andSOX2, along with differentiation and tumorigenic capabilities inmouse xenograft models of human SOC. High expression levelsof ALDH, but not CD133, correlated with spheroid formation

CSC marker expression and tumor forming capability. In clin-ical specimens of SOC, high levels of ALDH1A1 correlated withadvanced stage and poor prognosis. Pharmacologic or geneticblockade of ALDH blocked cell proliferation and reducedexpression of SOX2, the genetic ablation of which abolishedspheroid formation, whereas SOX2 overexpression inhibitedALDH1A1 expression and blocked spheroid proliferation. Tak-en together, our findings illustrated a new method to culturehuman ovarian CSC, and they defined a reciprocal regulatoryrelationship between ALDH1A1 and SOX2, which impactsovarian CSC proliferation and malignant progression. CancerRes; 76(1); 150–60. �2015 AACR.

IntroductionEpithelial ovarian cancer (EOC) accounts for the majority of

ovarian malignancies, and is one of the major causes of cancer-related death in women (1). Although improved surgical reduc-tion followed by standard combination chemotherapy is gener-ally initially effective, a majority of patients suffer from cancer

recurrence, which is less responsive to the original chemotherapy(2). Combined with the lack of reliable methods for diagnosis atthe early stages of the disease, the overall prognosis of EOCremains dismal, with a 5-year survival rate of approximately50% (3).

EOC is classified into several distinct histologic subtypes (4).Among these, serous ovarian carcinoma (SOC) is one of the mostcommon types of EOC, and there is clinical urgency to understandthe specific biologic features of SOC that can be exploited for itstreatment.

Recently, the concept of cancer stem-like cell (CSC) hasemerged as a promising theory that can explain the malignantbehavior of cancer, including chemo-resistance and metastasis(5). It is postulated that the refractory nature of EOC can beexplained by the existence of CSCs, as the vast intratumoralheterogeneity observed in EOC suggests that there is a smallpopulation of cells that survive initial therapy and repopulatefor later recurrence (6, 7). This hypothesis has driven theattempts to isolate CSCs from EOC to characterize their bio-logic nature.

Many markers for ovarian CSCs have been reported (8, 9).Pioneering works have identified tumorigenic populationsfrom ovarian cancer based on the expression of the surfacemarkers including CD44 (10, 11), CD117 (11), CD133 (12),CD24 (13, 14), or on the differential Hoechst-dye efflux (sidepopulation; ref. 15). These variations in CSC markers mayreflect the heterogeneous nature of CSCs from SOC. Alterna-tively, it is possible that experimental variations, such assources of CSCs (immortalized cell lines or passaged primary

1Department of Obstetrics and Gynecology, Niigata University Grad-uate School of Medical and Dental Sciences, Niigata, Japan. 2Divisionof Cancer Differentiation, National Cancer Center Hospital, Tokyo,Japan. 3Central Animal Division, National Cancer Center Hospital,Tokyo, Japan. 4Division of Molecular and Cellular Medicine, NationalCancer Center Hospital, Tokyo, Japan. 5Department of Pathology,National Cancer Center Hospital, Tokyo, Japan. 6Department ofObstetrics and Gynecology, Kitasato University Hospital, Kanagawa,Japan. 7Department of Gynecology, National Cancer Center Hospital,Tokyo, Japan. 8Division of Carcinogenesis and Cancer Prevention,National Cancer Center Research Institute, National Cancer CenterHospital, Tokyo, Japan.

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

T. Ishiguro and A. Sato contributed equally to this article.

Current address for H. Tsuda: Department of Pathology, National DefenseMedical College, Saitama, Japan; and current address for T. Kasamatsu, Depart-ment of Gynecology, Tokyo Metropolitan Bokutoh Hospital, Tokyo, Japan.

Corresponding Author: Koji Okamoto, National Cancer Center Research Insti-tute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan. Phone: 81-3-3542-2511; Fax:81-3-3542-2530; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-15-0361

�2015 American Association for Cancer Research.

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cells), growth conditions, or recipient mice, led to differences inthe published markers.

From recent studies in which tumor-initiation capability wasdirectly validated from fresh tumor samples, two markers, ALDHactivity and CD133 expression, have emerged as potent biomar-kers that can enrich cells with tumorigenic capability fromovariantumors (12, 16–18). The same studies reported that ALDH-positive cells were detectable in most of the ovarian tumorsexamined (16–18).

In fact, ALDH can be used as a CSC marker for a variety ofcancers (19). Especially, ALDH1A1 is amajor isoform responsiblefor the ALDH activity of CSCs (19), and hence one of thepromising candidates for CSC markers of ovarian cancer as wellas other cancers.

Considering the implied importance of ALDH in ovarianCSCs, it will be crucial to maintain and analyze ALDH-positiveCSCs in vitro. Although high levels of ALDH activity are asso-ciated with CSC-related features in ovarian cancer cell lines(20), there are no reports demonstrating the proliferation ofALDH-positive CSCs in in vitro culture, suggesting that there aresome unknown technical difficulties for their maintenanceunder in vitro conditions.

Previously, we demonstrated that the inhibition of Rho kinase(ROCK) markedly improved the maintenance of a spheroidculture of colon CSCs in vitro (21). The improved efficiency ofthe spheroid formation by ROCK inhibition was associated withthe sustained expression of CD44 variants (CD44v), which areknown to be among the established CSCmarkers of various typesof cancer (21).

In the present study, we found that ROCK inhibition wasessential for the spheroid formation from surgical specimen ofovarian cancer, and the established spheroids showed variouscharacteristics of CSCs. We investigated functional roles of stemcell-related markers for the maintenance of stemness of ovarianCSCs.

Materials and MethodsSpheroid culture

All procedures were performed under the protocol approvedby the Ethics Committee and the Animal Care and Use Commit-tees of the National Cancer Center or Niigata University. Tumorsamples were minced and dissociated with 150 U/mL collagenaseplus 50 U/mL hyaluronidase (Stemcell Technologies), and sequen-tially filtered through 100 mm, 70 mm, and 40 mm cell strainers(BD Falcon). After lysis of red blood cells with Red Blood CellLysis Solution (Miltenyi Biotec), the filtered cells were grown onultra-low-attachment culture dishes (Corning) in STEMPRO hESCSFM (Gibco) supplemented with 8 ng/mL bFGF (Invitrogen),penicillin/streptomycin, 20 mmol/L Y27632 (Wako), and 10 mg/mL insulin (Roche). For serial passage, spheroid cells were disso-ciated with Accumax (Innovative Cell Technologies) once every 10days. Xenograft establishment after transplantation of spheroidcells was performed as previously described (21).

Western blot analysesWestern blot analyses were performed as previously described

(21). Antibodies against SOX2, Nanog, CD44, CK7, ALDH1A1,actin, and CD133 were purchased from Cell Signaling Technol-ogy, Abcam, Thermo Scientific, Dako, Santa-Cruz Biotechnology,Sigma-Aldrich, and Miltenyi, respectively.

Immunohistochemical analysesImmunostaining of spheroid cells, mouse xenograft, or prima-

ry tumors was performed as previously described (21). Forimmunostaining of clinical specimen, SOC samples were stainedwith anti-ALDH1A1 (ab52492, Abcam), and the extent of thestaining was visually evaluated on a scale of 0 (no staining) to 3(strong staining). At least 1,000 cancer cells, not including stromalcells, were evaluated, and the mean value for each staining wascalculated.

In vitro assays for spheroid cellsCell growth or apoptosis was quantified by CellTiter-Glo Assay

(Promega) or Caspase-Glo 3/7 Assay (Promega). Limiting dilu-tion assays and qRT-PCR assays were performed as previouslydescribed (21).

Flow cytometry analysesDissociated single spheroid cells were filtered, incubated with

DAPI (BD Pharmingen) for the exclusion of nonviable cells, andused for ALDEFLUOR assays (StemCell Technologies Inc.) orstaining with the anti-CD133 antibody (CD133/1, conjugatedwith PE, Miltenyi). Samples were analyzed using a FACS Aria IIICell Sorter (BD Biosciences; ref. 21).

Lentivirus-mediated shRNA transductionLentivirus plasmids that express ALDH1A1 shRNAs (sh2:

TRCN0000276399, sh3: TRCN0000276459), SOX2 shRNAs(sh1: TRCN0000323281, sh5: TRCN0000359770), or controlshRNA were purchased from Sigma. SOX2-expressing lentiviralplasmid, pSin-EF2-SOX2-pur (22), was purchased fromAddgene. Preparation of virus-containing supernatants, virusinfection, and selection of infected cells were performed aspreviously described (23).

Luciferase assayDissociated spheroid cells were transfected using the neon

transfection system (Thermo Fisher Scientific), and luciferaseactivity was measured using dual-luciferase assay system (Pro-mega). For construction of ALDH1A1-luc, the promoter regionspanning �697 to þ315 was inserted into the KpnI-NcoI site ofpGL3-Basic (Promega).

Gene set enrichment analysesMicroarray analyses were performed using Agilent Whole

Human Genome 8 � 60 K Oligo microarrays (24). Microarraydata were accessible through Gene Expression Omnibus database(accession number GSE64999). Gene set enrichment analyseswere performed by using the gene set collection of version 3.7 ofthe Molecular Signatures Database (MSigDB).

Statistical analysesFor statistical analyses of spheroid cell experiments, either

Welch t test or Student t test was performed based on the resultsof F test. �, P < 0.05; ��, P < 0.01; ��, P < 0.001. Statistical analysesof clinical samples were performed using the GraphPad Prismver.4. (GraphPad Software Inc.). Univariate survival analysis wasperformed using the Kaplan–Meier method, and the significanceof difference between groups was examined using the log-ranktest. Multivariate survival analysis was carried out using the Coxproportional hazards regression model.

The Establishment of ALDH-Dependent Ovarian CSCs

www.aacrjournals.org Cancer Res; 76(1) January 1, 2016 151




ResultsSpheroid cells derived from human ovarian cancer showcharacteristics of CSCs

Recently, we established a spheroid culture of colonCSCs in thepresence of ROCK inhibitor, Y27632 (21). We attempted toestablish spheroid cultures of ovarian CSCs from surgical speci-mens by applying similar methods. We were able to establishlong-term spheroid culture in 5 out of 16 cases examined (Sup-plementary Table S1). Long-term spheroid culture was possibleonly in the presence of insulin/Y27632, suggesting that theseadditives conveyed an advantage for spheroid growth. Hematox-ylin and eosin (H&E) staining of the spheroids showedmorphol-ogy reminiscent of floating aggregates of serous ovarian cancer inascites (Fig. 1A; Supplementary Fig. S1A; ref. 25). The morpho-logic resemblance may reflect functional similarity with CSCs, aswe could establish the spheroid culture from cancerous ascites butnot from primary tumors in three cases (Supplementary Table S1,SOC-#4, #5, #9).

Next, we examined CSC-related features of the establishedspheroid cells. Serially diluted cells (#4) were used for tumorformation experiments upon transplantation into immuno-compromised NOG mice. In agreement with previous studies

(16), 1 � 104 cells were capable of forming xenograft tumors inthree out of six cases after the injection into mammary pad orflank (Supplementary Table S2). Another spheroid (#5) was alsocapable of forming xenograft tumors in all 16 cases, in which1 � 106 cells were injected at the flank of the recipient mice(data not shown). Immunostaining with Pax8, CK7, CA125, orHE4 (26), revealed that the formed xenograft tumors wereindistinguishable from the original tumors (Fig. 1B; Supplemen-tary Fig. S1B and S1C). Spheroid cells underwent differentia-tion and showed epithelial-like morphology if grown attachedin the presence of serum (Fig. 1C). Expression of ALDH1A1 andthe stem cell–related factors (Nanog and SOX2) was reduced,whereas that of the differentiation marker (CK7) was inducedat the protein level (Fig. 1D; Supplementary Fig. S1D).

The reduction of ALDH1A1 and SOX2 after differentiation wascorroborated at the RNA level (Fig. 1E; Supplementary Fig. S1E).Gene set enrichment analyses revealed that undifferentiatedspheroid cells preferentially express stem cell–related genes ofbreast cancer (Fig. 1F; ref. 27), whichmay reflect similarity of geneexpression profile of ovarian and breast CSCs (28, 29). Collec-tively, these results indicate that the spheroid cells show char-acteristics of CSCs.

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Figure 1.Spheroid cells derived from human ovarian cancer show CSC characteristics. A, top, bright-phase image of the indicated spheroids. Scale bars, 100 mm.Bottom, H&E staining of the spheroids. Scale bars, 20 mm. B, immunostaining of xenograft tumor, 5 months after transplantation (#2). Scale bars, 20 mm.C, bright-phase images of undifferentiated spheroid cells (#2 and #4) and the cells grown under differentiation conditions. Scale bars, 100 mm. D, Western blotanalyses of the cells shown in C. E, qRT-PCR analyses of the spheroid cells (#2, #4). F, gene set enrichment analyses of gene expression profiles betweenundifferentiated and differentiated cells.

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Cancer Res; 76(1) January 1, 2016 Cancer Research152




Expression of CSC/stem cell–related factors is induced byROCK inhibition in ovarian spheroids

Next, we examined the spheroid proliferation in the presence orabsence of insulin and/or Y27632, and found that the spheroidproliferation was markedly inhibited in the absence of Y27632but not insulin in two out of these three spheroids we examined(#2 and #5, Fig. 2A and B). The positive effect of Y27632 on theproliferation of the #4 spheroid was not clear over one week(Fig. 2A and B), but extended culture over one month revealedpreferential proliferation (>10-fold) in the presence of Y27632(data not shown). The inhibition of the proliferation in theabsence of Y27632 was associated with an increase in caspaseactivity (Fig. 2C), indicating that enhanced apoptotic cell deathcontributes to the growth inhibition.

In contrast to the colon CSCs (21), CD44v levels were notaugmented by Y27632 in any of the ovarian spheroid cells(Fig. 2D). Instead, two CSC markers (ALDH1A1 and CD133)and stem cell–related regulators (SOX2, Nanog, and Oct-3/4)were induced by the ROCK inhibitor in #2 and #5, but not in #4(Fig. 2D). In thepresence of ROCK inhibitor, a substantial fractionof #2 and #5 spheroid cells showed high levels of ALDH activityand CD133 (Fig. 2E and F), and RNA levels of ALDH1A1 andSOX2 were significantly higher in #2 and #5 than in #4 (Fig. 2G).

Thus, enhanced spheroid proliferation by the ROCK inhibitor in#2 and #5 was correlated with the robust induction of ALDH1,CD133, and the stem cell markers. The overall expression profileof #2 and #5 was in striking contrast with #4, which expressedhigh levels of CD44v but showed hardly detectable expression ofALDH1A1 or CD133 (Fig. 2D and G).

High levels of ALDHexpression are associatedwithCSC-relatedcharacteristics of ovarian spheroids

Recent studies indicate that ALDH and CD133 can enrich cellswith tumorigenic capability from ovarian tumors (12, 16–18).Hence, we focused on spheroid cells with high levels of ALDHactivity and CD133 expression (#2 and #5). First, we purifiedspheroid cells that express either high or low/undetectable levelsof ALDH activity (ALDHhigh and ALDHlow) by flow cytometry(Supplementary Fig. S2A). The ALDHhigh cells expressed higherlevels of the stem cell–related markers (SOX2, Oct-3/4, andNanog) than ALDHlow cells (Fig. 3A). The ALDHhigh cells weremore efficiently capable of proliferating as spheroids thanALDHlow cells (Fig. 3B and C). Levels of caspase activity in theALDHlow cells were higher than those in ALDHhigh cells (Supple-mentary Fig. S2B). Furthermore, ALDHhigh cells, but not ALDHlow

cells, could generate xenograft tumors in immuno-compromized

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Figure 2.High levels of ALDH expression are maintained by ROCK inhibition and associated with proliferation ovarian spheroids. A, time course analyses of cell growthof the indicated spheroids in the presence or absence of Y27632 and/or insulin. B, bright-phase images of spheroids. Scale bars, 100 mm. C, caspase assaysof spheroid cells. The #2 spheroid cells were used for normalizing caspase activity. D,Western blot analyses of the spheroids. E, FACS analyses of ALDH activity afterALDEFLOUR staining in the presence (left) or absence (left) of diethylaminobenzaldehyde (DEAB). F, FACS analyses of CD133 expression after staining withcontrol IgG or anti-CD133 antibody (AC133). G, qRT-PCR analyses of ALDH1A1 and SOX2 in the presence of Y27632þinsulin.






mice (Fig. 3D) with histologic features similar to the originaltumor (Supplementary Fig. S1C).

Next, we purified spheroid cells that express either high or low/undetectable levels of CD133 (CD133high andCD133low) by flowcytometry (Supplementary Fig. S2C). There was no significantdifference in expression of ALDH1A1 and the stem cell–relatedfactors (Supplementary Fig. S2D) or in spheroid formation (Sup-plementary Fig. S2E). The CD133high cells proliferated slowerthan CD133low cells (Supplementary Fig. S2F).

In order to determine whether CD133 expression affects theenrichment of ovarian CSCs by ALDH expression, the ALDHhigh/CD133high, ALDHlow/CD133high, ALDHhigh/CD133low, andALDHlow/CD133low cells from the #5 spheroids were purified byFACS sorting (Fig. 3E), and used to examine CSC-related features.In agreement with Fig. 3B and C, ALDHhigh/CD133high andALDHhigh/CD133low cells, but not ALDHlow/CD133high andALDHlow/CD133low cells, were capable of efficiently formingspheroids (Fig. 3F), and the cells with higher ALDH activityproliferated faster than those with lower ALDH activity (Fig.3G). Serial dilution assays indicated that the spheroid formationefficiencies of the ALDHhigh/CD133high and ALDHhigh/CD133low

cells were comparable, andwere significantly higher than those of

the ALDHlow/CD133high or ALDHlow/CD133low cells (Supple-mentary Table S3).

Of note, the ALDHhigh/CD133low cells rather grew faster thanthe ALDHhigh/CD133high cells in spheroid culture in vitro(Fig. 3G). In accordance, although both cells were capable offorming tumors in mouse xenograft assays, the tumors formedafter injection of the ALDHhigh/CD133low cells were bigger thanthose of the ALDHhigh/CD133high cells (Fig. 3H), suggesting thatCD133 marks cells with slower proliferation late. The fasterproliferation of the ALDHhigh/CD133low cells may not be attrib-uted to the difference in reduced apoptotic cell death as these cellsshowed similar caspase activities (Supplementary Fig. S2G).

ALDH1 expression is associated with high tumorigenicity andpoor prognosis in human serous ovarian cancer

Next, we compared ALDH1A1 immunostaining of the spher-oid cells with that from the original cancer. In accordance withdata from FACS analyses (Fig. 2E), the ALDH1A1 immunos-taining showed the expression of ALDH1A1 in #2 and #5, butnot in #4 (Fig. 4A, right). The staining of the original tumorindicated a similar tendency for ALDH1A1 expression (Fig. 4A,left).

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Examination of the ALDH1A1 staining in early and advancedstage SOC (Supplementary Table S4) revealed that ALDH1A1expression was markedly augmented at the advanced stage(Fig. 4B). Statistical studies of the 90 cases of advanced stagehigh-grade SOC indicated that although there was no associationbetween ALDH1A1 expression and cilinicopathologic parameters(Supplementary Table S5A), high levels of ALDH1A1 expressionwere associatedwith poor prognosis (Fig. 4C andD).Multivariateanalyses of the clinical data indicated that ALDH1A1 stainingserved as an independent prognostic factor (Supplementary TableS5B). Thus, in agreement with the meta-analyses of the previousstudies (30), high levels of ALDH1A1 expression are associatedwith cancer progression and poor prognosis.

ALDH activity is required for SOX2 expression andproliferation of ovarian CSCs

Next, we investigated functional importance of ALDH activityfor ovarian CSCs. Disulfiram (DS), an ALDH inhibitor, was usedto block ALDH activity in #2 and #5 that express high levels ofALDH. The DS treatment caused partial inhibition of ALDH

activity in both spheroids (Fig. 5A; Supplementary Fig. S3A).Nevertheless, the ALDH inhibition almost completely blockedthe formation and proliferation of spheroids (Fig. 5B and C;Supplementary Fig. S3B and S3C), and induced apoptotic celldeath (Fig. 5D).Of note, the inhibition of ALDHactivity led to thereduction of SOX2 expression (Fig. 5E; Supplementary Fig. S3D),while suppression of the other stemmarkers, Oct-3/4 and Nanog,was not clear (data not shown). The DS treatment did notsignificantly affect the expression of SOX2 RNA (Fig. 5F; Supple-mentary Fig. S3E). Presumably the reduction of SOX2 was notattributed to the accelerated apoptotic cell death of SOX2-expres-sing cells, because caspase inhibitors did not restore SOX2 expres-sion in the presence of DS, while they suppressed caspase acti-vation (Fig. 5G; Supplementary Fig. S3F) and restored cell via-bility (Supplementary Fig. S3F).

ALDH1A1 mediates SOX2 expression and proliferation ofovarian CSCs

In order to determine whether ALDH1A1 is responsible for theproliferation and SOX2 expression, we inhibited ALDH1A1

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Figure 4.ALDH1 expression is associated with advanced clinical stages and poor prognosis in human serous ovarian cancer. A, immunostaining of ALDH1A1 of the indicatedspheroids (right) and the correspondingprimary tumors (left). Scale bars, 50mm.B, top, representative immunostainingofALDH1A1 (score0–3). Bottom, distributionof ALDH1A1 expression in early (FIGO stages I and II; n ¼ 23) and advanced (FIGO stages III and IV; n ¼ 90) stages of SOCs. Horizontal lines, average values.C and D, Kaplan–Meier analyses of overall (C) and progression-free (D) survival of patients suffering from the advanced stages of SOCs. The patients were stratifiedinto high (n ¼ 62; solid lines) and low (n ¼ 28; dotted lines) expression groups according to ALDH1A1 expression.






expression by shRNA lentiviral transfer (Fig. 6A). Although theshRNA introduction caused apparent inhibition of ALDH1A1in both #2 and #5 (Fig. 6B and Supplementary Fig. S4A),significant ALDH activity was still observable in #5, but notin #2 after the inhibition of ALDH1A1 (Fig. 6C; SupplementaryFig. S4B), suggesting that other isoforms contribute to ALDHactivity in #5.

Microarray data indicated that, in addition to ALDH1A1, therewere several ALDH isoforms that were expressed in #5 with levelsrelatively higher than in #2 or #4 (Supplementary Fig. S4C). Ofthose,Western blot analyses of four isoforms (1A2, 3A2, 5A1, and18A1) revealed that ALDH1A2 and ALDH3A2 were expressed inthe ALDHhigh cells at levels higher than in the ALDHlow cells(Supplementary Fig. S4D). Thus, these isoforms may also con-tribute to the ALDH activity in #5.

The inhibition of ALDH1A1 blocked the formation and pro-liferation of the spheroids (Fig. 6D and E; Supplementary Fig. S4Eand S4F), induced apoptotic cell death (Fig. 6F), and suppressedspheroid forming efficiency (Supplementary Table S6), indicatingthat ALDH1A1 was required for the survival and proliferation ofALDH-positive CSCs. In accordance with SOX2 suppression byDS (Fig. 5E; Supplementary Fig. S3D), the inhibition of ALDH1A1suppressed SOX2 expression in both spheroids (Fig. 6B; Supple-mentary Fig. S4A). It is likely that the suppression of SOX2 byALDH1A1 was mainly caused at the posttranscriptional level,

because DS treatment or the inhibition of ALDH1A1 caused onlymodest effects on SOX2 RNA expression (Fig. 5F; SupplementaryFigs. S3E and S4G).

The proliferation of ovarian CSCs is regulated throughreciprocal regulation by ALDH1A1 and SOX2

Because SOX2 expression was associated with ALDH-depen-dent spheroid proliferation (Figs. 5E and 6B; SupplementaryFigs. S3D and S4A), we examined whether the proliferation ofthe spheroid cells was also dependent on SOX2. shRNA-mediatedinhibition of SOX2 (Fig. 7A; Supplementary Fig. S5A) markedlysuppressed formation and proliferation of spheroids (Fig. 7B andC; Supplementary Fig. S5B and S5C), and spheroid formingefficiency (Supplementary Table S7), while the inhibition aug-mented apoptotic cell death (Fig. 7D). Thus, SOX2, as well asALDH1A1, is required for the survival and proliferation of ovarianCSCs.

In order to determine whether elevated levels of SOX2 affectthe proliferation of the ovarian CSCs, we introduced exogenousSOX2 into the #5 spheroid cells via lentivirus-mediated genetransfer (Fig. 7E). Unexpectedly, SOX2 overexpression caused areduced proliferation (Fig. 7F) and enhanced apoptotic celldeath (Supplementary Fig. S5D). In the presence of DS, SOX2overexpression further reduced proliferation and spheroid for-mation (Fig. 7F and G).

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Figure 5.ALDH activity is required for SOX2 expression and proliferation of ovarian CSCs (#5). A, FACS analyses of ALDH activity in the presence or absence of DS (2 daysafter the treatment). B, bright-phase images of spheroids (4 days after the DS treatment). Scale bars, 100 mm. C, time course of cell growth of the spheroids.D, caspase activity of the DS-treated spheroid cells. E, Western blot analyses of the spheroids at 2 days after the treatment with the indicated amounts of DS.F, qRT-PCR analyses of ALDH1A1 and SOX2 in the spheroids. G, Western blot analyses of the DS-treated spheroid cells in the presence or absence of caspaseinhibitors. The cells were preincubated with 100 mmol/L zVAD-FMK (Abcam), 25 mmol/L DEVD-CHO (Calbiochem), or DMSO for 2 hours and incubated for2 days in the presence or absence of 20 mmol/L DS.

Ishiguro et al.





Notably, SOX2 overexpression totally abolished ALDH1A1expression (Fig. 7E), which is likely to contribute to the inhi-bition of the CSC proliferation. SOX2 overexpression in the #4cells also resulted in the suppression of the proliferation andALDH1A1 inhibition (Supplementary Fig. S5E and S5F). SOX2regulated the transcriptional levels of ALDH1A1, because theSOX2 overexpression markedly inhibited ALDH1A1 RNAexpression (Fig. 7H; Supplementary Fig. S5G) and suppressedthe promoter activities in transient luciferase assays (Fig. 7I).Collectively, the elevated expression of SOX2 transcriptionallyblocks ALDH1A1 expression while ALDH1A1 is required forSOX2 expression.

DiscussionROCK is involved in regulation of cytoskeletons via the phos-

phorylation of its targets (31), and can affect proliferation ofnormal stem cells and CSCs: its inhibition facilitates proliferationof embryonic stem cells via suppression of dissociation-inducedapoptosis (32, 33) and of colon CSCs via induction of CD44v(21).Here, we demonstrated that ROCK inhibition also promotesproliferation of a subset of ovarian CSCs through induction ofALDH1A1 and the stem cell–related factors including SOX2.However, in contrast to colon CSCs, ROCK inhibition-dependent

proliferation in ovarian CSCs was not associated with CD44vinduction (Fig. 2D).

These discoveries led to the establishment of an in vitroculture of ovarian CSCs that are positive for ALDH1A1 andCD133. In contrast to the #2 and #5 spheroids, the #4 spher-oids showed high levels of CD44v but negligible levels ofALDH1A1 and CD133. Considering that CD44 is reported asanother CSCmarker (10, 11), there may be two types of ovarianCSCs: one is positive for ALDH and CD133, and the other forCD44.

The investigation of ALDH-positive spheroid cells revealed thatthese cells meet critical criteria for CSCs, including spheroidformation, the expression of stem cell markers, and the potentialto differentiate, and most importantly, the ability to form tumorsthat are histologically identical to the original primary tumor. Onthe other hand, CD133-positive cells showed rather slower pro-liferation both in vitro and in vivo. Previous reports suggest that theALDHþ/CD133þ cells represent cells with tumor-forming capa-bility (17, 18). The difference may be attributed to the heteroge-neity of the clinical sample, the difference in anti-CD133 anti-bodies used for the experiment, or subtle differences in experi-mental conditions.

Meta-analyses of recent studies indicated that elevated ALDHexpression was associated with poor prognosis in patients with

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Figure 6.ALDH1A1 is required for SOX2 expression and proliferation of ovarian CSCs (#5). A, an experimental scheme. B, Western blot analyses after infection of theindicated lentiviruses. C, flow cytometry analyses of ALDH activity of the infected cells. D, bright-phase images of the infected cells. Scale bars, 100 mm.E, time course of the proliferation of the infected cells. F, caspase activity of the infected cells (day 2).






ovarian cancer (30, 34–37), although some reports indicateotherwise (38). Our data on immunostaining of high-grade SOCdemonstrated that higher levels of ALDH1A1 are associated withan advance stage and poor prognosis, warranting the furtherinvestigation of ovarian cancer and CSCs that are positive forALDH.

SOX2 plays an essential role in CSCs (39–41) and associatedwith the malignant progression of ovarian cancer (42). Func-tional analyses by gene knockdown and a chemical inhibitorrevealed that ALDH and SOX2 are essential for the proliferationof ovarian CSCs. Of note, both ALDH1A and SOX2 expressioncontribute to chemo-resistance of ovarian cancer cell lines(35, 43–45). Therefore, SOX2 and ALDH may mediate itscapability to confer chemo-resistance to ovarian CSCs as wellas stem cell–related features.

Remarkably, our data revealed that expression of ALDH1A1and SOX2 affected each other: ALDH1A1 was required for SOX2expression, whereas SOX2 overexpression caused ALDH1A1 inhi-bition. These observations, combined with the essential roles ofthese regulators in spheroid proliferation, suggest an existence of afeedback regulation for CSC proliferation, in which SOX2 andALDH1A1 form a negative feedback loop (SupplementaryFig. S5H). The potential feedback regulation between SOX2 andALDH1A1 may be beneficial for the survival and proliferation ofovarian CSCs due to reduced perturbations of levels of theseregulators.

In humans, ALDH family is composed of 19 isoforms withsimilar catalytic functions (46). Although ALDH1A1 is regardedas a major form responsible for ALDH activity (19), other iso-forms (ALDH1A3, ALDH3A2, and ALDH7A1) are also overex-pressed in ovarian cancers (47). In fact, our data indicated thatALDH1A2 and ALDH3A2, in addition to ALDH1A1, may con-tribute to ALDH activity in #5. Future studies will clarify thepotential contribution of these isoforms to ovarian CSCs.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: T. Ishiguro, H. Ohata, Y. Ikarashi, T. Enomoto,K. Tanaka, K. OkamotoDevelopment of methodology: T. Ishiguro, A. Sato, H. Ohata, R.-u TakahashiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Ishiguro, A. Sato, M. Yoshida, T. OndaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T. Ishiguro, H. Ohata, R.-u Takahashi, M. Yoshida,T. EnomotoWriting, review, and/or revision of the manuscript: T. Ishiguro, A. Sato,H. Ohata, Y. Ikarashi, M. Yoshida, H. Tsuda, T. Onda, T. Kato, T. Enomoto,H. Nakagama, K. OkamotoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): T. Ochiya, M. Yoshida, H. Tsuda, T. Kato,T. Kasamatsu, H. NakagamaStudy supervision: T. Enomoto, K. Tanaka, H. Nakagama, K. Okamoto

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Figure 7.Reciprocal regulation of ALDH1A1 and SOX2 are involved in control of CSC proliferation. A, Western blot analyses of the spheroid cells (#5) after infection ofcontrol lentiviruses or the viruses expressing SOX2 shRNAs. B, bright-phase images of the infected spheroid cells. Scale bars, 100 mm. C, time course ofproliferation of the cells shown in B. D, caspase activity of the infected cells (day 2). E, Western blot analyses of SOX2-introduced cells. F, time courseof the proliferation of the infected cells in the presence or absence of DS. G, bright-phase images of the cells shown in F (4 days after DS treatment).H, qRT-PCR analyses of ALDH1A1 in cells shown in E. I, Inhibition of the ALDH1A1 promoter by SOX2 in transient reporter assays. The indicated vectors werecotransfected into #5 for 48 hours and luciferase assays were performed.

Ishiguro et al.





AcknowledgmentsThe authors thank Hiroaki Sakai, Rui Uchino, and Katsuhide Ikeda for

technical assistance and Takashi Kohno and Hitoshi Ichikawa for help withthe genome analyses.

Grant SupportThis research was supported by a Grant-in-Aid for Scientific Research in

Innovative Areas fromMEXT (K. Okamoto), a Grant-in-Aid for Cancer Researchfrom the Foundation for Promotion of Cancer Research (K. Okamoto), a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion ofScience (JSPS; K. Okamoto), a Research Resident Fellowship from Grant-in-Aid

for Young Scientists (B) from JSPS (H.Ohata), National Cancer Center Researchand Development Fund (25-A-2, 25-B-6; K. Okamoto and H. Ohata), a HealthLabour Sciences Research Grant from The Ministry of Health, Labour andWelfare (K. Okamoto and H. Ohata), and a Grant-in Aid from Takeda ScienceFoundation (H. Ohata).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 4, 2015; revised September 14, 2015; accepted October 2,2015; published OnlineFirst December 15, 2015.

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Ishiguro et al.




2016;76:150-160. Published OnlineFirst December 15, 2015.Cancer Res Tatsuya Ishiguro, Ai Sato, Hirokazu Ohata, et al. Cancer Stem-like Cells with an Enhanced Proliferative Capacity

Model of OvarianIn VitroEstablishment and Characterization of an

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