Cancer Letters 339 (2013) 237–246

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Cancer Letters

journal homepage: www.elsevier .com/locate /canlet

Ovarian tumor initiating cell populations persist following paclitaxeland carboplatin chemotherapy treatment in vivo

0304-3835/$ - see front matter � 2013 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.canlet.2013.06.014

⇑ Corresponding author at: Vincent Center for Reproductive Biology, Departmentof Vincent Obstetrics and Gynecology, Massachusetts General Hospital, Boston, MA02114, USA. Tel.: +1 617 724 2825; fax: +1 617 726 0561.

E-mail address: brueda@partners.org (B.R. Rueda).

Kashmira Kulkarni-Datar a,b, Sandra Orsulic c, Rosemary Foster a,b, Bo R. Rueda a,b,⇑a Vincent Center for Reproductive Biology, Department of Vincent Obstetrics and Gynecology, Massachusetts General Hospital, Boston, MA 02114, USAb Harvard Medical School, Boston, MA 02115, USAc Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA

a r t i c l e i n f o

Article history:Received 24 April 2013Received in revised form 6 June 2013Accepted 10 June 2013

Keywords:Ovarian cancer tumor initiating cellsSca-1 cellsCD133 cells

a b s t r a c t

Development of recurrent platinum resistant disease following chemotherapy presents a challenge inmanaging ovarian cancer. Using tumors derived from genetically defined mouse ovarian cancer cells,we investigated the stem cell properties of residual cells post-chemotherapy. Utilizing CD133 and Sca-1 as markers of candidate tumor initiating cells (TIC), we determined that the relative levels of CD133+

and Sca-1+ cells were unaltered following chemotherapy. CD133+ and Sca-1+ cells exhibited increasedstem cell-related gene expression, were enriched in G0/G1-early S phase and exhibited increased tumorinitiating capacity, giving rise to heterogeneous tumors. Our findings suggest that residual TICs may con-tribute to recurrent disease.

� 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Ovarian cancer is the leading cause of death among women withgynecological cancers in the United States. It is estimated that in theyear 2013 alone 22,240 women will be diagnosed with ovarian can-cer and �14,030 women will succumb to the disease [1,2]. Non-symptomatic onset, delayed diagnosis, and the aggressive natureof ovarian cancer make clinical management especially challenging.The primary treatment modality for women presenting with ad-vanced stage ovarian carcinoma often includes surgical debulkingimmediately followed by taxane and platinum based chemotherapy[3–6]. While most patients respond to first line therapy, with time asignificant number will develop highly aggressive recurrent diseasethat is often platinum resistant [3,7]. This resurgence is a majorobstacle to successful long-term disease management in these wo-men since the treatment options for recurrent and/or platinum resis-tant ovarian cancer are currently limited and less effective [8],reviewed in [9,10]. Thus, understanding the underlying biology ofthe chemoresistant tumor cells that potentially lead to tumor resur-gence could provide a significant advantage in therapeutic manage-ment of recurrent ovarian cancer.

Development of recurrent disease has been attributed, at leastin part, to a unique population of cells often referred to as can-cer stem cells (CSC), tumor initiating cells (TIC), cancer initiating

cells (CIC) or tumor repopulating cells reviewed in [11,12]. Thispopulation, in the majority of the cases, is believed to be rela-tively rare within the larger tumor bulk and is thought to haveregained the capacity to both self-replicate and give rise to moredifferentiated cells resulting in a hierarchal heterogeneous tumor[13–16], reviewed in [11,17]. Unlike rapidly dividing differenti-ated tumor cells, TICs often evade the toxicity of standard che-motherapeutics due to their slow division rate and the abilityto take advantage of efflux properties associated with multidrug-resistance [18,19–26]. TICs have been identified and character-ized in leukemia and several solid cancers including breast,lung, prostate, brain, liver and ovarian [15,27–30,32,33] reviewedin [11]. Enrichment or isolation of ovarian TICs has been success-fully performed via multiple strategies including dye exclusionvia efflux properties. Ovarian TICs were enriched in a side popu-lation (SP) fraction that was identified by its ability to effluxHoechst 33342 dye [19,22–24,27–31]. Alternatively, differentialexpression of specific cell surface antigens or differential enzy-matic activity has been utilized in the isolation andcharacteriza-tion of TICs. Expression of CD44, CD133, and CD117 andaldehyde dehydrogenase (ALDH) enzymatic activity either aloneor in combination have been utilized in the identification, isola-tion and functional testing of TICs in human ovarian tumors,ascites and ovarian cancer cell lines [13,21,32–38]. Somein vitro and in vivo studies provide evidence to suggest there isan enrichment of ovarian cancer stem-like cell populationspost-chemotherapy [18,19,23,25,26,29,38] supporting thehypothesis that these cells resist conventional cytotoxic thera-pies and contribute to the development of recurrent disease.

238 K. Kulkarni-Datar et al. / Cancer Letters 339 (2013) 237–246

Our objective was to utilize tumors derived from a geneticallydefined mouse ovarian cancer cell line to assess the role of putativeovarian TICs in tumor resurgence. The T2 cell line was generatedfrom poorly differentiated mouse ovarian tumors formed followinginjection of p53

�/�; c-myc; myristoylated-Akt1 expressing mouse

ovarian surface epithelial cells [39]. Subsequent injection of T2cells in immunocompromised mice leads to rapid formation ofpoorly differentiated tumors. We used this in vivo experimentalsystem to model tumor resurgence following treatment with pac-litaxel and carboplatin. We determined whether tumor cell sub-populations identified by expression of specific stem cell markerspersist post-chemotherapy, are capable of inducing tumor forma-tion and display stem cell-like properties. Our results suggest thatsuch TICs are present within the T2-derived tumors and possiblycontribute to the resurgence of tumor growth.

2. Materials and methods

2.1. T2 cell culture

The mouse ovarian cancer cell line T2 (p53�/�

; c-myc; myristoylated-Akt1) wasmaintained at subconfluency in Dulbecco’s modified Eagles medium (DMEM,Mediatech Inc.) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA)and 1% penicillin–streptomycin (Gibco Carlsbad, CA) and passaged 2 times a weekas previously described [39].

2.2. T2 tumor generation

T2 cells (2 � 106) were resuspended in PBS:Matrigel� (BD Biosciences, Bedford,MA) in 1:1 ratio and injected subcutaneously into 6–8 week old female NOD/SCIDmice (Jackson Laboratory, Bar Harbor, ME). Animals were monitored regularly to as-sess the formation and growth of tumor. Tumor formation was typically observed4–6 weeks after injection. Animals with excessive tumor burden or in a moribundstate were euthanized. All animal experiments were conducted in accordance withthe recommendations in the Guide for the Care and Use of Laboratory Animals ofthe National Institutes of Health. The protocol was approved by the MassachusettsGeneral Hospital Sub-Committee on Research and Animal Care.

2.3. Treatment with chemotherapeutic agents

Mice bearing tumors (200–500 mm3) were randomized into two cohorts. Micein the vehicle cohort received Cremophor: ethanol (1:1, paclitaxel vehicle) and sal-ine (carboplatin vehicle) once a week by intraperitoneal (i.p.) injection. Mice in thepaclitaxel and carboplatin cohort received paclitaxel (15 mg/kg) (Sigma–Aldrich, St.Louis, MO) and carboplatin (50 mg/kg) (Hospira, Chicago, IL) once a week by i.p.injection. Tumors were measured twice weekly using calipers and tumor volume(mm3) was determined using the formula [length (mm) �width (mm) �width(mm)]/2. For each animal, the percent change in tumor volume was determinedby comparing tumor volume over the course of treatment to the starting tumor vol-ume. Mouse weights were measured twice weekly to assess potential toxic effectsof chemotherapeutics. At the end of the treatment period, all remaining animalswere euthanized and tumors were harvested. Portions of the tumor were used forflow cytometric analyses and fixed in paraformaldehyde for histological analysis.

Table 1Tumorigenic capacity of serially diluted dual negative, Sca-1+, CD133+ and dualpositive cells transplanted in vivo.

Cells 5 � 103 5 � 102 5 � 10

Dual negative 3/3 (21–31) 1/3 (56) 0/3Sca-1+ 3/3 (7–21) 3/3 (28–64) 2/3 (35–70)CD133+ 3/3 (14–21) 3/3 (28–45) 2/3 (30–64)Dual positive N/A 1/1 (21) 1/1 (35)

Sorted dual negative, CD133+, Sca-1+and dual positive cells were injected subcu-taneously into NOD/SCID mice at the indicated cell number and tumor formationwas monitored. Numbers in () indicates range of days required for tumor formation.

2.4. Flow cytometry

T2 tumor tissue was harvested and dissociated into a single cell suspension aspreviously published [36]. Briefly, tissue was chopped and incubated in a mixture ofDNAse I and collagenase II with agitation at 37 �C. Following enzymatic dissocia-tion, mechanical disruption was performed in a Stomacher�. Dissociated cells werewashed in HBSS buffer (HBSS + 2% FBS + 1 mM EDTA) and treated with ACK solutionfor red blood cell lysis. Cells were further spun on a ficoll gradient to eliminatewhite blood cells and cellular debris. The residual cells were resuspended in coldPBS buffer (PBS + 2% FBS + 1 mM EDTA) and stained with Livedead� cell viabilitydye (Invitrogen, Carlsbad, CA), CD31-FITC (BD Biosciences, Bedford, MA), CD45-FITCand CD133-PE or Sca-1-APC or both (Miltenyi Biotec, Bergisch Gladbach, Germany)for 10 min at 4 �C and washed to remove excess antibody. Stained cells were resus-pended in PBS buffer and were sorted on a BD FACSAria cell sorter or fixed with 4%paraformaldehyde post-staining and analyzed within 24 h on a BD LSRII analyzer.Re-analysis of sorted populations determined purity to be greater than 93%. Analy-sis was performed using the FlowJo 7.6.5 software (Treestar, Inc., Ashland, OR).

2.5. Cell cycle analysis

Chemotherapy naïve T2 tumors were dissociated and cells were stained withanti-CD31-FITC, anti-CD45-FITC and anti-CD133-PE and/or anti-Sca-1-APC antibod-ies as described in the previous section. Cells were further stained with Vybrant�

Dyecycle violet stain for DNA content analysis according to the manufacturer’sinstructions. Verapamil (100 lM) was added to cells for Dyecycle violet dye reten-tion. Cells were maintained at 37 �C until evaluated on a BD LSRII analyzer. Cell cy-cle analysis was performed using the FlowJo 7.6.5 software (Treestar, Inc., Ashland,OR).

2.6. Sca-1 and CD133 cell sorting and reinjection

Chemotherapy naïve tumors originally derived from the T2 cell line were har-vested. Viable single-cell suspensions were stained with APC-conjugated Sca-1and/or PE-conjugated CD133 antibodies. CD133+ Sca-1+, CD133+ Sca-1�, CD133-

Sca-1+ and CD133� Sca-1� cells were sorted on a BD FACSAria. Isolated cell popula-tions were serially diluted as indicated in Table 1, resuspended in 1:1 PBS:Matrigel�

and re-injected subcutaneously into female NOD/SCID mice. A portion of the cellswas snap frozen for RNA analysis.

2.7. Histology

Formalin fixed paraffin-embedded (FFPE) tissue pieces were used for histologi-cal analysis. Tissue sections of 5 lm thickness were dewaxed, rehydrated andstained with hematoxylin and eosin (H&E) to assess histology. Images were cap-tured using a Nikon TE2000-S microscope and SPOT software (Diagnostic Instru-ments, Inc., Sterling Heights, MI).

2.8. Immunocytochemistry

Five micron FFPE tissue sections were dewaxed, rehydrated and analyzed for E-cadherin expression. Antigen retrieval was carried out using 10 mM sodium citrate,pH 6.0. Tissues were blocked with PBS buffer containing 10% goat serum, 1% BSAand 0.1% Triton X-100 for 1 h at room temperature. Sections were incubated over-night at 4 �C with E-cadherin antibody (SC-7870, Santa Cruz biotechnology, SantaCruz, CA) diluted 1:100 dilution in blocking buffer. The sections were then washedwith PBS and incubated with Alexa Fluor 568 conjugated donkey anti-rabbit (Invit-rogen, Carlsbad, CA) secondary antibody (1:200) for 1 h at room temperature. Nu-clei were stained with DAPI and an appropriate dilution of rabbit IgG was used asa negative control. Fluorescent images were captured using a Nikon Ni-U uprightepiflouresence microscope and NIS-Elements software (NIKON instruments, Inc.,Melville, NY).

2.9. Real-time PCR analysis

RNA was isolated from frozen sorted Sca-1� CD133�, Sca-1+ CD133�, Sca1�

CD133+, and Sca-1+ CD133+ cells using the RNeasy plus micro kit (Qiagen, Valencia,CA) following the manufacturer’s instructions. Two micrograms of isolated RNAwere used to synthesize cDNA using the Superscript™ VILO kit (Invitrogen, Carls-bad, CA). DNA amplification was carried out using CFX96 (BIO-RAD, Hercules,CA). Detection was performed by measuring the fluorescence of SYBR Green dyebound to dual-stranded DNA. The real-time thermal cycle program was 95 �C for30 s followed by 40 cycles of 95 �C for 5 s and 58 �C for 30 s. Primers and probesfor mouse Sca-1, CD133, Sox2,Oct4, Nanog, Tcf-1, Gli1, Hes1 and beta-actin genes weredesigned in house (Supplementary Table 1) and were purchased from Invitrogen(Carlsbad, CA). The levels of target gene mRNA were normalized to the internal con-trol gene beta-actin. The relative expression of each gene was compared to the genelevels in autologous CD133� Sca-1� cells and the fold change in expression levelwas determined by the 2

(�DDct)method.

K. Kulkarni-Datar et al. / Cancer Letters 339 (2013) 237–246 239

2.10. Statistical analysis

Non-parametric Wilcoxan rank sum tests for unpaired samples were used tocompare tumor sizes in the vehicle and paclitaxel and carboplatin treatment exper-iments. Significance was set at p 6 0.05. STATA (College Station, TX) v10 softwarewas used for analysis. Paired samples were compared using a paired t-test with sig-nificance set at p 6 0.05.

3. Results

3.1. Paclitaxel and carboplatin treatment inhibits growth of T2 tumors

The effect of paclitaxel in combination with carboplatin on T2tumor growth in vivo was assessed by administering either vehicleor paclitaxel and carboplatin to mice bearing T2-derived tumors.Although mice treated with vehicle alone exhibited a significant(p < 0.03) increase in tumor volume over the course of the treat-ment period, tumor volume was relatively unchanged in mice trea-ted with paclitaxel and carboplatin (Fig. 1A, p = 0.126). Thesignificant difference in tumor volume (p < 0.03) observed betweenthe vehicle treated and the paclitaxel and carboplatin treatedgroups demonstrated that paclitaxel and carboplatin were effec-tive in impeding T2 tumor growth in mice.

To determine if the withdrawal of paclitaxel and carboplatinwould result in tumor resurgence, treatment was discontinued

Fig. 1A. Paclitaxel and carboplatin treatment impedes ovarian tumor progression. Mice bor paclitaxel and carboplatin (T/C). Change in tumor volume is shown relative to the tumrepresent the standard error of the mean (SEM). *p < 0.03, #p < 0.05 (Wilcoxon rank sum

Fig. 1B. Resurgence of ovarian tumor growth following withdrawal of T/C treatment. Mdelivered on day 14 and change in tumor volume post- T/C withdrawal was assessed. Chaexperiment. Error bars represent the S.E.M.

and subsequent changes in tumor volume were monitored. Uponcessation of paclitaxel and carboplatin treatment, a dramatic in-crease in tumor volume was observed relative to tumor volumeat the end of treatment (Fig. 1B). These results suggest that whilepaclitaxel and carboplatin treatment effectively impedes T2 tumorgrowth, it does not completely eliminate all viable tumor cellswhich persist in sufficient number to repopulate the tumor.

3.2. Sca-1 and CD133 expressing cells persist post-chemotherapy

TICs in solid tumors have been identified based on the expres-sion of cell surface markers such as CD117, CD133, CD44, andCD24 as well as by differential ALDH activity [32,33,35,36,40–43]. Therefore, to determine whether the T2-derived tumor cellsthat persist following paclitaxel and carboplatin treatment com-prise a sub population of candidate TICs, we initially analyzedthe expression of several TIC markers in chemotherapy naïve T2derived tumors (data not shown). Since the T2 cell line is derivedfrom mouse ovarian surface epithelium [39], we carried out analy-sis of both the murine specific cell surface protein stem cell anti-gen-1 (Sca-1, also known as Ly6A/E) expression and markersreported to be expressed on human ovarian TICs. Sca-1 is knownto enrich for stem cell populations in murine models of leukemia,prostate cancer and in mammary gland tumors [40–46]. We

earing tumors derived from T2 cell line (T2 tumors) were treated with either vehicleor volume at the beginning of the experiment. N = 6–8 animals per group. Error bars

test).

ice bearing T2 tumors were treated with either vehicle or T/C. Last dose of T/C wasnge in tumor volume is shown relative to the tumor volume at the beginning of the

240 K. Kulkarni-Datar et al. / Cancer Letters 339 (2013) 237–246

determined that CD133 and Sca-1 were expressed in T2 derived tu-mors, with CD133+ and Sca-1+ cells comprising 0.4–1.8% and 2.0–6.0% of the total viable cell population (data not shown). These rel-atively low levels of CD133+ or Sca-1+ expressing cells in T2 tumorsare consistent with levels of TICs reported in other studies[21,36,38,47]. Unlike other studies showing CD117 and CD44 levelsin primary or xenograft tumors, the expression of these markerswas inconsistent (data not shown). We therefore focused our ef-forts on examining CD133+ and Sca-1+ expressing cells in the T2 tu-mors [26,38].

CD133 and Sca-1 expression was then analyzed in tumors de-rived from mice treated with vehicle or paclitaxel and carboplatinfor 14 days. As shown in Fig. 1A, paclitaxel and carboplatin treat-ment effectively suppressed growth of T2-derived tumors. Surpris-ingly, flow cytometric analysis of tumors post-chemotherapeutictreatment revealed that the level of cells expressing CD133 orSca-1 did not differ significantly between the paclitaxel and carbo-platin and vehicle treated samples (Fig. 2A and B; #p = 0.2749,*p = 0.9028) despite the dramatic difference in tumor volume.

3.3. Sca-1+, CD133+ and CD133+Sca-1+ cells have increased tumorinitiating capacity and give rise to heterogeneous tumors

To determine the tumor initiating capacity of CD133+ and Sca-1+ cells, T2 tumors were dissociated into a single cell suspensionwhich was sorted to generate CD133� Sca-1� (dual negative),CD133+Sca-1� (CD133+), CD133�Sca-1+ (Sca-1+) and an extremely

Fig. 2. CD133+ and Sca-1+ cells persist in tumors post-chemotherapy. Mice bearingT2 tumors were treated with vehicle or T/C for 14 days. Post-treatment tumorswere analyzed for expression of (A) CD133 or (B) Sca-1 by flow cytometry. Errorbars represent the S.E.M. #p = 0.2749, *p = 0.9028 (student’s t test). Significance wasset at p 6 0.05.

rare CD133+Sca-1+ (dual positive) cell fractions (Fig. 3A). Serialdilutions of the sorted populations were injected subcutaneouslyinto NOD/SCID mice and tumor formation was assessed over a2 month period (Table 1). More rapid tumor formation was ob-served following injection of CD133+, Sca-1+ or dual positive cellscompared to tumor formation following injection of cells that didnot express either marker. Tumor formation was observed in miceinjected with as few as 50 CD133+, Sca1+ or dual positive cells. Incontrast, injection of 5000 dual negative cells was required to gen-erate tumors with latency similar to that observed following injec-tion of 50 dual positive cells. These results suggest that cellsexpressing Sca-1 and CD133 have enhanced tumor-initiatingcapacity compared to cells that express neither marker.

The histology of the tumors generated from the isolatedCD133+, Sca-1+ and dual positive cell populations was similar tothe poorly differentiated phenotype of the parent T2 tumor(Fig. 3B–E). Since CD133 expression marks both epithelial andmesenchymal stem cells [48], we assessed the expression of theepithelial cell marker E-cadherin in tumors generated followinginjection of CD133+, Sca-1+ or dual positive cells (Fig. 3F–H). Weobserved abundant expression of E-cadherin suggesting that theT2 tumor derived Sca-1+ and CD133+ cells are capable of generat-ing epithelial tumors.

Recent studies in several cancer types, including ovarian, havedetermined that highly purified populations of TICs are capableof recapitulating a heterogeneous tumor phenotype suggestive ofa hierarchical arrangement of cells within tumors [11,35]. Wetherefore assessed the CD133 or Sca-1 cell surface marker pheno-type of tumors that formed following injection of purifiedCD133+ or Sca-1+ cells (Fig. 4A and B). Tumors that formed follow-ing injection of a purified CD133+ cell fraction comprised bothCD133+ and CD133� cells. Likewise, tumors generated from iso-lated Sca-1+ cells comprised both Sca-1+ and Sca-1� fractions. Col-lectively, these data indicate that CD133+ and Sca-1+ cells exhibitthe potential to give rise to more differentiated cells and can reca-pitulate tumor heterogeneity.

3.4. Sca-1+, CD133+ and CD133+Sca-1+ cells are enriched in the G0/G1

phase of the cell cycle

Tumor initiating cells are postulated to replicate infrequentlyand thereby escape the cytotoxic effects of chemotherapeutics thattarget actively dividing cells. We expected that fewer T2 tumor-de-rived TICs would be in S or G2/M phase of the cell cycle when com-pared to the cell cycle distribution of the total cell population. Toinvestigate this hypothesis, single cell suspensions generated fromchemotherapy naïve T2 derived tumors were stained with the DNAbinding dye-Dye Cycle violet and the DNA content of dual negative,CD133+, Sca-1+ and dual positive cells was assessed by FACS(Fig. 5A and B). As shown, the relative percentage of cells in theG0/G1-early S phase of the cell cycle was lowest in the dual nega-tive cells, which had minimal tumor initiating capacity. In contrast,nearly all of the highly tumorigenic dual positive cells were in G0/G1-early S phase while the percentage of Sca-1+ or CD133+ cells inG0/G1-early S was intermediate (Fig. 5B). These data suggest thatthe Sca-1+, CD133+ and dual positive cell fractions comprise morenon-replicating quiescent G0/G1-early S cells and the percentageof these cells in each sub-population correlates with its relative tu-mor initiating capacity.

3.5. CD133+ and Sca-1+ cells express stemness associated genes anddisplay differential activation of developmental pathways

Previous studies suggest that TICs are characterized by in-creased expression of genes associated with an undifferentiatedphenotype [25,26,48]. We analyzed the expression of Oct4, Nanog

Fig. 3. Tumors derived from CD133+, Sca-1+ and dual positive cells express epithelial cell marker, E-cadherin and maintain parent tumor histology. (A) T2 tumors werestained and FACS sorted into CD133+, Sca-1+ and dual positive populations. Isolated populations were reinjected subcutaneously into NOD/SCID mice. The histopathology of(B) unsorted T2 tumors and (C–E) tumors generated from CD133+, Sca-1+ and dual positive sorted cell fractions were analyzed using hematoxylin and eosin staining. (F–H)E-cadherin (red) expression was detected in tumors generated from CD133+, Sca-1+ and dual positive cells using immunocytochemistry. DAPI (blue) was used forvisualization of nuclei. White scale bars = 100 lm and black scale bars = 50 lm.

K. Kulkarni-Datar et al. / Cancer Letters 339 (2013) 237–246 241

and Sox2 in CD133+ and Sca-1+ T2 tumor derived cells using quan-titative real-time PCR (Fig. 6A–E). Analysis of CD133 and Sca-1expression in the sorted populations verified abundant expressionof the CD133 transcript in CD133+ and dual positive cells (Fig. 6A)and high Sca-1 gene expression in the Sca-1+ and dual positive cells(Fig. 6B). As expected, neither gene was expressed in the dual neg-ative cell fraction. Elevated levels of Oct4 transcripts were observedin the CD133+ and dual positive cells, relative to Oct4 expression indual negative cells (Fig. 6C). Lower levels of Nanog were observedin cells expressing either CD133 or Sca-1 alone as well as in thedual positive cells relative to expression in CD133�Sca-1� cells(Fig. 6D). While Sox2 transcript levels did not differ between cellsexpressing Sca-1 and the dual negative cells, elevated levels ofSox2 transcript were observed in CD133+ and dual positive cellsalthough this difference was not significant. These results suggestthat expression of genes associated with an undifferentiated phe-notype are distinctly expressed in T2 tumor derived cell popula-tions distinguished by differential CD133 and Sca-1 expression.

We also investigated activation of the Notch, Hedgehog andWnt/bcatenin signaling pathways in the sorted cell populations.These pathways are active during early development and are be-lieved to be involved in the maintenance of TIC in an undifferenti-ated state [18,22,30,49]. We analyzed expression of the pathwayspecific downstream transcription factors Hes1 (Notch), Gli1

(Hedgehog) and Tcf-1 (Wnt/b-catenin). Expression of these factorsis often increased in TICs and that increase is indicative of pathwayactivation in the cells. While no difference in the levels of Gli-1 orHes-1 was observed between dual negative cells and Sca-1+,CD133+ or dual positive cells, we observed significant increase inTcf-1 transcript levels in CD133+ and dual positive cells (p < 0.05;Fig. 6F–H) relative to Tcf-1 levels in dual negative cells. These re-sults suggest differential activation of the Wnt/b-catenin develop-mental pathway in the T2 tumor derived TICs.

4. Discussion

The research focus in a greater majority of cancers has ex-panded to include an emphasis on an often elusive and potentiallyrare population of tumor cells believed to possess stem cell-likeproperties. The specific properties of cancer stem cells are, to somedegree, context specific. However, most researchers would agreethat common CSC properties include the capacity to self replicate,the ability to give rise to more differentiated daughter cells and rel-ative quiescence, and chemoresistance when compared to theirmore differentiated counterparts. Mounting evidence supportsthe existence of ovarian CSC, which led us and others to postulatethat these cells contribute to the resurgence of ovarian cancer post-chemotherapy.

Fig. 4. Tumors generated from CD133+ cells or Sca-1+ cells exhibit both TIC and non-TIC populations. (A) Tumors generated from CD133+ cells were analyzed using flowcytometry to determine the expression of CD133+ TIC cells and CD133� cells. Unstained cells from CD133+ cells derived tumors were used as controls. (B) Tumors generatedfrom Sca-1+ cells were analyzed using flow cytometry to determine the expression of Sca-1+ TIC cells and Sca-1� cells. Unstained cells from Sca-1+ cells derived tumors wereused as controls. Percentages represented by each population are indicated in the gates.

Fig. 5. CD133+, Sca-1+ and dual positive cells represent slow-cycling, quiescent populations. (A) Chemotherapy naïve T2 tumors were analyzed for the expression of CD133+,Sca-1+ and dual positive cells (B) DNA dye, DyeCycle Violet, was used to determine the DNA content and the cell-cycle status (G0/G1– Early S and Late S/G2–M phases) of thedual negative, CD133+, Sca-1+and dual positive cells (n = 3). Error bars represent the S.E.M.

242 K. Kulkarni-Datar et al. / Cancer Letters 339 (2013) 237–246

Using immunocompromised mice bearing tumors generatedfollowing injection of the mouse T2 ovarian cancer cell line, wehave shown that tumor growth is restricted by treatment with pac-litaxel and carboplatin in vivo. We have obtained similar resultswith human ovarian tumor xenografts [50] although the response

of T2-derived tumors to chemotherapy was less dramatic withtumor volume being held in check rather than reduced over thetreatment period. The significance of this limited response is notyet known but it may be due to the T2 tumor genotype (p53

�/�;

c-myc; myristoylated-Akt1) which could confer some resistance

Fig. 6. CD133+, Sca-1+ and dual positive cells differentially express markers associated with stem cells. The expression of (A) CD133, (B) Sca-1, (C) Oct 4, (D) Nanog, (E) Sox2, (F)Gli1, (G) Hes1 and (H) Tcf-1 was analyzed by real-time PCR in dual negative (DN), CD133+, Sca-1+ and dual positive (DP) cells. The levels of each gene were normalized to theinternal control beta-actin. Fold change in transcript levels are shown relative to transcript levels in the dual negative cells. Error bars represent the S.E.M. *p < 0.03, #p < 0.05(student’s t test). Significance was set at p 6 0.05.

K. Kulkarni-Datar et al. / Cancer Letters 339 (2013) 237–246 243

to paclitaxel and carboplatin based chemotherapy and result in astable disease pattern. Amplifications of Akt and myc are relativelyfrequent events in high-grade ovarian carcinomas [51–54].Overexpression of AKT in vitro has been associated with increasedincidence of platinum resistant disease [55–57].

We demonstrated that cessation of paclitaxel and carboplatintreatment resulted in a dramatic resurgence of T2-derived tumorgrowth. We hypothesize that this increase in tumor growth follow-ing treatment depends in part on the presence of CSC-like cells inthis model.

244 K. Kulkarni-Datar et al. / Cancer Letters 339 (2013) 237–246

We have identified a sub-population of cells expressing CD133and Sca-1 in T2 cell line derived tumors that display CSC proper-ties. Interestingly, T2 cells failed to exhibit CD133 or Sca-1expression in vitro (data not shown). There was little to no differ-ence in the percentage of CD133+ and Sca1+ cells in tumors de-rived from mice treated with paclitaxel and carboplatin relativeto levels in tumors from mice treated with vehicle despite signif-icantly lower tumor volumes. This is in agreement with previousstudies in lung cancer xenograft models which showed no de-crease in the levels of CD133+ tumor cells following cytotoxictherapy [58]. Other investigators have suggested that stem cellnumber may actually increase in response to chemotherapy[59,60]. Although we did observe someincrease in CD133+ andSca-1+ cells in a subset of experiments, those changes were notstatistically significant. Our data are consistent with the hypoth-esis that conventional chemotherapy effectively eliminates rap-idly proliferating differentiated tumor cells. There was noenrichment, however, of TICs in the bulk tumor post-chemother-apy, with the ratios of TICs to non-TICs remaining the same. Thepersistence of TICs following chemotherapy suggests that thesecells can contribute to resurgent tumor growth. To determinewhether the CD133+ or Sca-1+ T2 tumor derived cells are capableof forming tumors in vivo and display properties associated withstem cells, we performed a series of experiments to assess theirdifferential tumor initiation capacity, cell cycle status and expres-sion of genes associated with an undifferentiated multi-potentcell phenotype. Tumor initiation assays in which purifiedpopula-tions of T2 tumor derived CD133+ Sca-1

�, CD133�Sca-1+ or

extremely rare CD133+Sca-1+ cells were injected intoimmunocompromised NOD/SCID mice suggest that expression ofeither marker is sufficient to confer tumorigenicity. Silva et al. re-cently reported the increased tumorigenicity of dual positiveALDH+CD133+ human ovarian cancer cell populations comparedwith the individual ALDH+ or CD133+ cell populations in suggest-ing a further enrichment of CSC in the dual population [35]. Sim-ilarly, we determined that the dual positive CD133+Sca-1+ T2tumor derived cells are particularly tumorigenic when comparedto CD133+Sca-1

�or CD133

�Sca-1+ cells. Since the dual positive

population is extremely rare we were unable to repeat the tumor-ogenecity assay and additional investigation may be required toestablish their tumorogenic capacity. We observed that theCD133

�Sca-1

�dual negative cells generate tumors following injec-

tion of high cell numbers but this capacity was lost as the in-jected cell number was decreased. It is likely that thistumorigenic capacity is due to the presence of either residualCD133+ or Sca-1+ cells since we did not obtain 100% purity ofour sorted populations or TICs defined by unknown surface mark-ers. Finally, the tumors generated following injection of purifiedcell populations mimicked the poorly differentiated phenotypeof the T2 parent tumor and expressed high levels of the epithelialmarker E-cadherin. These resultsconfirm that CD133 and Sca-1expression mark T2 tumor derived TIC capable of generating epi-thelial tumors. This is in contrast to published studies suggestingthat in several cancers CD133+ cells may play a role in epithelialto mesenchymal transition [48].

CD133+ and Sca-1+ populations display stem cell-like features inother tumor types or tumor models, including endometrial, pros-tate, breast and ovarian [36,43,46,61]. Cell cycle analysis of T2 tu-mor derived dual negative, CD133+, Sca-1+ and dual positive cellfractions revealed that a greater percentage of CD133+, Sca-1+

and dual positive cells are in the G0/G1-early S phase of the cell cy-cle and presumably represent a resting phase population. Our re-sults concur with previous studies in human and mouse modelswhich have shown enrichment of CD133+ and Sca-1+ cells in theG0 phase of the cell cycle [43,62]. While the enrichment in theG0/G1-early S phase is less pronounced in the T2 tumor derived

CD133+ cells, we believe these cells may represent cells that areslowly turning over and giving rise to the other populations. Dueto the relatively rare occurrence of the CD133+ and extremely raredual positive cells, it is likely difficult to determine the absolutecell cycle distribution of these populations. Collectively, our resultssuggest that the CD133+, Sca-1+ and the dual positive populationsare relatively quiescent, a hallmark of CSC.

Previous studies have demonstrated the expression of severalgenes associated with pluripotency in CSC isolated from tumors[25,26,48]. We analyzed the expression of Oct4, Nanog and Sox2in the four isolated cell populations. An increase in expressionof Oct4 was observed in the CD133+, Sca-1+ and dual positivepopulations compared with expression in the CD133

�Sca-1

�

cells, suggesting the Oct4 expressing cell fractions share anundifferentiated phenotype. Interestingly, we did not seeincreased Nanog or Sox2 expression in any T2 tumor derived cellpopulation that expressed CD133 or Sca-1 suggesting a higherpotential in these cells for pre-disposed differentiation to acertain cell type [63]. In addition, we analyzed the activationstatus of the developmental and stem cell signaling pathwaysNotch, Hedgehog and Wnt/beta-catenin in the isolated T2 tumorderived cell fractions. There was no difference in activation ofNotch and Hedgehog pathways between dual negative cellsand cells expressing either or both CD133 and Sca-1 cell surfacemarkers. This observation suggests that Notch and Hedgehogsignaling potentially contribute to the maintenance of proliferat-ing non-TICs in the T2 model of ovarian cancer. Interestingly,we detected significantly higher expression of Tcf1 factor inCD133+and dual positive T2 tumor derived cells suggesting thatactive Wnt/b-catenin signaling may be involved in maintenanceof the these TIC populations.

In conclusion, our study provides evidence to suggest that T2tumor derived cells that express CD133+ and Sca-1+ are TIC thatpersist following paclitaxel and carboplatin chemotherapy.Although further investigation is required to delineate the specificcontribution of TICs to tumor regeneration and the development ofplatinum resistance, our data indicate that adjuvant therapy tar-geting TICs in combination with standard regimens may provebeneficial in the managing ovarian cancer.

Funding

This research was funded in part by grants from the AdvancedMedical Research Foundation (BRR) and the Vincent Memorial Re-search Funds (BRR).

Conflict of interest

None declared.

Acknowledgements

The authors would like to thank Laura Prickett-Rice, Kat Folz-Donahue and Meredith Weglarz at the HSCI Flow Cytometry CoreFacility for their help with cell sorting and cell cycle analysis;and colleagues at the VCRB laboratories for critical review of themanuscript.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.canlet.2013.06.014.

K. Kulkarni-Datar et al. / Cancer Letters 339 (2013) 237–246 245

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