CD44v8-10 Is a Cancer-Speciﬁc Marker for Gastric Cancer ... · Tumor and Stem Cell Biology...

Tumor and Stem Cell Biology

CD44v8-10 Is a Cancer-Specific Marker for Gastric CancerStem Cells

Wen Min Lau1, Eileen Teng1, Hui Shan Chong1, Kirsten Anne Pagaduan Lopez1, Amy Yuh Ling Tay2,Manuel Salto-Tellez1,3, Asim Shabbir2, Jimmy Bok Yan So2, and Shing Leng Chan1

AbstractThe surfacemarker CD44 has been identified as one of several markers associated with cancer stem cells (CSC)

in solid tumors, but its ubiquitous expression in many cell types, including hematopoietic cells, has hindered itsuse in targeting CSCs. In this study, 28 paired primary tumor and adjacent nontumor gastric tissue samples wereanalyzed for cell surface protein expression. Cells that expressed pan-CD44 were found to occur at significantlyhigher frequency in gastric tumor tissues.We identified CD44v8-10 as the predominantCD44 variant expressed ingastric cancer cells and verified its role as a gastric CSC marker by limiting dilution and serial transplantationassays. Parallel experiments using CD133 failed to enrich for gastric CSCs. Analyses of another 26 primary samplesshowed significant CD44v8-10 upregulation in gastric tumor sites. Exogenous expression of CD44v8-10 but notCD44 standard (CD44s) increased the frequency of tumor initiation in immunocompromised mice. Reciprocalsilencing of total CD44 resulted in reduced tumor-initiating potential of gastric cancer cells that could be rescuedby CD44v8-10 but not CD44s expression. Our findings provide important functional evidence that CD44v8-10marks human gastric CSCs and contributes to tumor initiation, possibly through enhancing oxidative stressdefense. In addition, we showed that CD44v8-10 expression is low in normal tissues. Because CD44 also marksCSCs of numerous human cancers, many of which may also overexpress CD44v8-10, CD44v8-10 may provide anavenue to target CSCs in other human cancers. Cancer Res; 74(9); 1–12. �2014 AACR.

IntroductionGastric cancer is a major cause of cancer-related death

worldwide, with low survival and high recurrence rates forpatients with advanced disease (1–3). Therefore, new therapiesfor treatment of gastric cancer are urgently needed. The cancerstem cell (CSC) hypothesis proposes that a specific subset ofCSCs is primarily responsible for initiating and maintainingtumor growth (4, 5), a proposition that may explain the highfrequency of relapse and resistance to current therapeuticmodalities that focus on reduction of tumor bulk withoutconsidering tumor heterogeneity. Definitive CSC markers forgastric cancer have yet to be characterized, although severalstudies have identified gastric CSCs from cell lines (6–9).

CD44 is a cell surface transmembrane glycoprotein encodedby the CD44 gene, a 20-exon DNA segment (10), of which exons1–5 and 16–20 are spliced together and translated into CD44s,the standard isoform. In addition, the variant exons 6–15 canbe alternatively spliced and assembled in different combina-tions with the standard exons to generate other variant(CD44v) protein isoforms. More recently, CD44 has beenrecognized as a CSC marker in several types of cancer (11).As CD44s is ubiquitously expressed in many cell types (12–16),its usefulness as a CSC marker may be limited. Furthermore,conflicting data in the field implicate CD44 in both tumorsuppression and progression (17–19), largely attributed to theexpression of alternatively spliced variants. In light of this, weinvestigated the role of CD44 splice variants in gastric cancer.In this study, we isolated gastric CSCs using two well-knownmarkers for CSCs, EpCAM and CD44. Further investigation ofthe expression of CD44 variants in gastric cancer revealed thatCD44v8-10 is specifically upregulated in tumor cells, and itsexpression confers an advantage in tumor initiation.

Materials and MethodsStudy approval

All patient tissue samples were collected with informedpatient consent from National University Hospital Singapore(Singapore) according to the Institutional Review Board guide-lines. All animal experiments were conducted with the approv-al of Institutional Animal Care and Use Committee in theNational University of Singapore.

Authors' Affiliations: 1Cancer Science Institute of Singapore; 2Depart-ment of Surgery, National University of Singapore, Singapore, Singapore;and 3Centre for Cancer Research & Cell Biology, Queen's UniversityBelfast, Belfast, Ireland, United Kingdom

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

W.M. Lau and E. Teng contributed equally to this work.

J.B.Y. So and S.L. Chan conceptualized the project.

Corresponding Author: Shing Leng Chan, Cancer Science Institute ofSingapore, 14Medical Drive, MD6, #12-01, Singapore 117599, Singapore.Phone: 65-65167211; Fax: 65-68739664; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-2309

�2014 American Association for Cancer Research.

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Isolation andpurificationof single cells fromdissociatedtumor tissue

Samples were washed twice in sterile Hank's Balanced SaltSolution (HBSS) before mincing finely with scalpel blades.Tissue pieces were incubated in 0.1% dithiothreitol/PBS toremove mucus and washed twice in HBSS before incubation at37�C for 2 hours in 0.2-mm sterile filtered DMEM/F12 media(Invitrogen) containing 100 U/mL hyaluronidase (H1136; Sig-ma), 300 U/mL collagenase type II (C6885; Sigma), 5 ng/mLhuman recombinant insulin (12585; Invitrogen), and 1 ng/mLhydrocortisone (H2270; Sigma). Digestionmixture was washedtwice in HBSS by centrifugation at 1,200 � g for 8 minutes.Pelletedmaterial was resuspended inHBSS and filtered using a40-mm cell strainer (Falcon) to collect single cells. Single-cellsuspensions were passed through LD and dead cell removalcolumns (Miltenyi Biotec) to remove cellular debris and deadcells. All procedures were carried out under sterile conditions.

Flow cytometric analysis and cell sortingSingle cellswerecountedwithahemocytometerusingTrypan

blue exclusion of dead cells. A total of 1 � 105 and 3 � 105 cellswere used for direct and indirect staining, respectively. For cellsisolated from clinical gastric samples, CD45þ blood cells andGlyAþ erythroid precursors were gated out and excluded fromanalysis (Supplementary Fig. S1). For xenograft-derived cells,murine H-2Kd MHC class I alloantigen-positive (H-2Kdþ) cellswere excluded from analysis. Cells were analyzed on a BD LSRIIflow cytometer (BD Biosciences). Median fluorescence intensity(MFI) values were calculated using FlowJo software (TreeStar).Cell sorting was performed on a BD FACSAria (BD Biosciences).Sytox Blue (S34857) from Invitrogen was used to exclude deadcells in all analyses. Post-sort analysis was performed to ensurethat purity of cell fractions was �90%. Cells were washed twicein sterile PBS and counted before resuspension in Matrigel (BDBiosciences) for xenograft transplant assays. Rat anti-humanCD44v8-10 specific antibody was kindly provided by Prof.H. Saya (Keio University, Tokyo, Japan) and was generated aspreviously described (20). Briefly, CD44v8-10 was overexpressedin McA RH-7777 rat epithelial cells that do not express anyendogenous isoforms of CD44. These cells were then used toimmunize rat host to raise CD44v8-10 antibodies. Other anti-bodies used are described in Supplementary Materials andMethods.

Patient-derived xenograft modelsSingle-cell suspensions orminced tissue pieces weremixed at

2:1with sterileHBSS/Matrigel (BDBiosciences) in afinal volumeof 200 mL per injection and administered subcutaneously. ForCD44 overexpression studies, vector control and CD44-trans-fected cells were injected onoppositeflanks of the same animalsto minimize experimental variability from differences in recip-ient mouse hosts. For cell-dosing experiments, 20 to 25,000sorted or unsorted cells were injected per site. Six- to 8-week-oldnonobese diabetic/severe combined immunodeficient (NOD/SCID) or NOD/SCID/IL2Rg� (NSG) mice were used for xeno-graft transplantations and anesthetized intraperitoneally with acocktail of xylazine (11 mg/kg) and ketamine (72 mg/kg). Micewere checked weekly for tumor formation and sacrificed when

tumors were 1.5 cm in diameter (usually 6–10 weeks) or after 20weeks (if no tumor was observed).

Primary gastric samples and cell linesPrimary gastric cancer cells used in our study include GC59,

GC71, GC101, GC121, and GC123. Established patient-derivedxenograft lines include GC16, GC21, GC38, GC45, GC84, andGC119. Primary cell line GC38-adh was established in vitrofrom GC38 xenograft line as described below. Other cell linesused were MKN28 (JCRB Cell Bank, Japan), SNU5 (AmericanType Culture Collection), and TMK1 (provided by Dr. PatrickTan, Duke-NUS, Singapore). These cell lines were authenticat-ed by 16-loci short tandem repeat (STR) profiling (LGC Stan-dards) in November 2013.

Primary cell cultureAll cultures were maintained in humidified 37�C incubators

supplemented with 5% CO2. Primary cell cultures were estab-lished from GC38 xenograft tumors. Tumor spheroids werefirst derived from freshly minced tumor pieces in a 10-cmtissue culture dish in serum-free low-glucose Dulbecco's Mod-ified Eagle Medium (DMEM; Invitrogen; details in Supplemen-tary Data). Spheroids were propagated by dissociation withTrypLE (Invitrogen) for 10 minutes at 37�C, centrifugation at1,200� g for 8minutes, and resuspension in PBS before passingthem through a cell strainer (30 mm) to remove dead cellaggregates. Live single cells were then replated in serum-freeDMEM at a density of 10,000 single cells per 35-mm dish. GC38tumor spheroids were EpCAMþ as characterized by flowcytometric analysis, with 55% to 85% CD44þ (EpCAMþCD44þ)cell populations. Established cultures of tumor spheroids wereused to derive adherent monolayer primary cultures of GC38cells. Spheroids were transferred to DMEM containing 10%FBS, which facilitated the growth of adherent cells after aperiod of 3 to 4 weeks. These adherent cells were passaged andexpanded as the GC38-adh primary cell line.

In vivo limiting dilution assaysNon-mouse cells from each xenograft were sorted on the

basis of the indicated surface phenotypes and injected subcu-taneously inNSGmice. Eachmouse received the same cell doseof indicated fractions from the same xenograft and was eitherharvestedwhen tumor generated from any fraction reached 1.5cm diameter (usually 6–10 weeks), or 20 weeks later. Mice wereconsidered negative for tumor formation when there was nopalpable tumor. For functional assays of CD44s and CD44v8-10overexpression, cells were sorted for the respective populationand injected into mice at indicated doses. The frequency ofcancer-initiating cells was calculated using theWeb-based toolExtreme Limiting Dilution Assay (ELDA; Walter and Eliza HallInstitute, Parkville VIC, Australia).

Cell proliferation assaysGC38-adh cells, stably transfected with pcDNA3 vector,

CD44s, or CD44v8-10, were fractionated for the relevant pop-ulation and 3,000 cells were seeded in each of five replicatewells in a 96-well plate. Cell proliferation over a 5-day periodwas measured using WST-1 cell proliferation reagent (Roche

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Diagnostics) according to the manufacturer's instructions.Briefly, 10 mL of WST-1 reagent was added per 100 mL ofculture medium and incubated for 2 hours before measure-ment of absorbance at 460 nm in a plate reader (Tecan).Absorbance readings were normalized against blank wellsand growth curves were plotted with data points showingmean � SD.

Depletion of CD44 in TMK1 cellsShort hairpinRNA (shRNA) that targets CD44and scrambled

control shRNA were obtained from Origene (TG314080). CD44shRNA consisted of two different shRNA sequences targetedagainst humanCD44 (NM_000610). Cellswere transfectedwithshRNA plasmids using Lipofectamine 2000 (Invitrogen) asdescribed above. For CD44 "rescue" experiments, TMK1 cellswere cotransfected with 6 mg each of CD44 shRNA plasmids(Origene) and either CD44s, CD44v8-10, or pcDNA3 vector asshRNA-resistant rescue constructs. These shRNA-resistantrescue constructs were engineered by mutating relevant resi-dues in the CD44 plasmid sequences targeted by the shRNAconstructs. The mutagenesis primers and shRNA sequencesare provided in Supplementary Table M1. Transfections wereperformed as described above. TMK1 cells were sorted by flowcytometry for the relevant populations before injecting theminto NSG mice for tumor formation assay.

ELISAELISA was performed using human CD44 ELISA kit

(Ab45912; Abcam). Conditionedmedia (100–300 mL) from cellscultured in 10-cm dishes was assayed according to the man-ufacturer's protocol.

HistopathologyTissue samples were fixed in 10% formalin and embedded in

paraffin before sectioning at 4-mm thickness. Hematoxylin andeosin (H&E; Sigma) staining was performed on all sections andevaluated by a pathologist.

Statistical analysisAll error bars represent the mean � SD. Paired two-tailed

Student t tests and Fisher exact test were performed usingGraphPad Prism (GraphPad Software). Chi-squared (x2) testswere performed using the Web-based software for ELDA(http://bioinf.wehi.edu.au/software/elda/; Walter and ElizaHall Institute of Medical Research; ref. 21). For all statisticaltests, P < 0.05 was considered to be significant. For flowcytometric data, MFI was calculated using FlowJo software(TreeStar) where relevant.

ResultsIdentificationofEpCAMandCD44as surfacemarkers forgastric CSCsA candidate approach was undertaken to identify putative

gastric CSCs from primary gastric cancer specimens becausevarious surface markers have previously been used to describeCSCs from different cancers. Cells from 28 paired primarytumor and adjacent nontumor tissues from patients with

gastric cancer (Supplementary Table S1) were isolated andanalyzed by flow cytometry for potential CSC markers includ-ing CD44, CD133, CD34, CD117, CD90, CD166, and EpCAM aswell as CD45 (hematopoietic cells), CD31 (endothelial cells),and CD140b (fibroblasts). Of these, gastric cancer cells werepositive for EpCAM, CD44, CD133, and CD166 expression. Inparticular, EpCAM levels were significantly higher in cells from28 gastric tumors compared with paired adjacent nontumortissue (P ¼ 0.0006; Fig. 1A). EpCAM is a well-establishedepithelial marker that is highly expressed in most carcinomasincluding gastric cancer (22, 23). Also, EpCAMþ but notEpCAM� cells initiated tumors in mice (Supplementary Fig.S1C), suggesting that gastric CSCs should be a subpopulationwithin the EpCAM-expressing tumor cells; hence, we usedEpCAM as an identifier for gastric tumor epithelial cells andto exclude nontumor cells.

Following this, flow cytometric analyses of EpCAM-expres-sing cells indicated the absence of CD90þ, CD34þ, and CD117þ

cells, and a high proportion of CD166þ cells (60%–70%) in bothtumor and nontumor sites. CD44- and CD133-expressing cellswere found in the range of 0.8% to 54% and 0.6% to 45%,respectively. Analyses of CD44þ or CD133þ cells in theEpCAMþ fraction isolated from 21 paired tumor and nontumorpatient samples revealed a consistently higher proportion ofCD44þ cells (P¼ 0.0124), but not CD133þ cells at the tumor site(P ¼ 0.861; Fig. 1B–D). Nevertheless, CD44 and CD133 werefurther analyzed as potential markers for gastric CSCs.

We established a robust gastric cancer xenograft model bysubcutaneous implantation of primary tumors taken frompatients with gastric cancer. Histologic analysis verified thatall xenograft tumors recapitulated the original primary tumors,as shown for GC16 (well-differentiated), GC21 (moderatelydifferentiated), and GC38 (poorly differentiated; Fig. 2A). Theproportion of EpCAMþCD44þ cells in GC16, GC21, and GC38xenografts was 3.6%, 58%, and 44%, respectively (Fig. 2B).Limiting dilution assays were performed to determine thefrequency of CSCs in EpCAMþCD44þ fractions from GC16,GC21, and GC38 xenografts (Table 1 and Fig. 2C). The esti-mated number of CSCs in EpCAMþCD44þ fractions wassignificantly enriched by 204-, 17-, and 8-fold in GC16, GC21,and GC38 xenografts, respectively, compared with EpCAMþ

CD44� fractions that were depleted of CD44þ cells. Parallelexperiments using EpCAMþCD133þ cells showed that gastricCSCs are not enriched in this fraction as both CD133þ andCD133� cells have similar tumor-initiating potential (Supple-mentary Table S2). Hence, we concluded that EpCAM andCD44 may be better surface markers for identifying gastricCSCs than CD133.

Serial transplantations were also performed by reisolatingEpCAMþCD44þ cells from first transplants in NSG mice andinjecting these cells into recipient mice as second transplants.The proportion of CD44þ cells in xenograft tumors of both firstand second transplants remained unchanged from the originaltumors (Fig. 2D). Similarly, xenograft tumors of both first andsecond transplants showed the same histology (Fig. 2E).Because the injected cells are �95% CD44þ, this suggests thatEpCAMþCD44þ cells are able to differentiate to reestablishtumor heterogeneity of the original patient tumor as well as to

CD44v8-10 Marks Gastric Cancer Stem Cells

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self-renew and sustain tumor growth in serially passagedxenografts. This also demonstrates that CD44þ cells retainedtumor-forming potential during serial transplantation. Takentogether, these data indicate that gastric CSCs are enriched inthe EpCAMþCD44þ fraction of gastric tumors.

CD44v8-10 is the predominant CD44 variant in gastriccancer xenografts and marks gastric CSCs

To determine the expression of CD44 variants in xenografttumors, PCR primers were designed in constant exons 5 and16/17, flanking the variable region of the CD44 gene (Fig. 3A).

PCR amplification of cDNA from xenograft tumors GC16,GC21, and GC38 generated two main products—324 and720-bp fragments whose sequences were verified to be CD44s,the standard transcript in many cell types, and CD44v8-10,a variant first identified in colon carcinomas, respectively(Fig. 3A; ref. 24). By quantitative PCR (qPCR) analysis, wefound that the level of CD44v8-10 transcript is highest in GC21,followed by GC38 and GC16 (Fig. 3B). Most importantly, levelsof CD44v8-10 transcripts correlated with the estimatednumber of gastric CSCs in the unsorted fractions (Fig. 2Cand Table 1).

Figure 1. EpCAM-expressing cellsin gastric cancer tissues are tumorepithelial cells containingsubpopulations of CD44þ andCD133þ cells. Flow cytometricanalyses of surface markerexpression in cells isolated from 28paired gastric tumors and adjacentnontumor tissue. A, EpCAM levelsin 28 gastric tumors comparedwithadjacent nontumor tissue. MFI wascalculated using FlowJo software(Supplementary Fig. S1B).Horizontal bars, the mean value.Patient clinical information isincluded in Supplementary TableS1. B, proportion of CD44þ andCD133þ cells in gastric cancercompared with adjacent nontumortissue. Representative flowcytometry data plots from 3patients with gastric cancer (GC16,GC59, and GC71) are shown.Proportion (%) of CD44þ orCD133þ cells within the EpCAMþ

population is shown in the top rightquadrant. C and D, proportion ofCD44 and CD133 cells in EpCAM-expressing cells isolated from 21paired tumor and nontumor sites.Cells were stained with antibodiesagainst EpCAM and CD44 orCD133 and analyzed by flowcytometry. Data were analyzed forstatistical significance usingStudent paired t test.

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We next used antibodies specific to CD44v8-10 to determinewhether the EpCAMþCD44þ cells identified in clinical samplesusing pan-CD44 antibody consist of CD44v8-10–expressingcells. The specificity of CD44v8-10 antibody was verified as

follows: first, the antibody binds to CD44v8-10 but not CD44swhen either of these was transiently expressed in MKN28,a gastric cancer cell line devoid of CD44 expression (Sup-plementary Fig. S2A). Second, the extracellular domain of

Figure 2. Gastric CSCs are enriched in EpCAMþCD44þ cell fractions. A, gastric cancer xenograft lines from three clinical biopsies of well-differentiated(GC16), moderately differentiated (GC21), and poorly differentiated (GC38) gastric cancer. Top, H&E staining of primary gastric cancer biopsies.Bottom, H&E staining of respective xenogeneic patient-derived tumors. Scale bars, 200 mm. B, proportion of CD44þ cells in respective xenograft lines.C, summary of limiting dilution assays estimating the frequency of CSCs in unsorted, EpCAMþCD44þ, and EpCAMþCD44� cell fractions from GC16,GC21, and GC38 xenografts. Differences between groups were tested for statistical significance using x

2 test (degrees of freedom ¼ 1; 95% confidenceintervals). �, statistically significant P values. Detailed data for limiting dilution analysis and estimation of gastric CSC frequency are shown in Table 1.D, flow cytometric analysis of EpCAMþCD44þ population in primary and secondary xenograft tumors from serial transplantation experiments generatedfrom sorted EpCAMþCD44þ cells. E, H&E staining showing histology and cellular composition of tumors initiated by EpCAMþCD44þ cells from first andsecond xenograft transplants. Scale bars, 100 mm.






CD44v8-10 (sol-CD44v8-10) but not sol-CD44s blocked thebinding of the CD44v8-10 antibody to gastric cancer cells(Supplementary Fig. S2B), indicating that binding of the anti-body to these cells is specific to the CD44v8-10 protein.

Using this CD44v8-10–specific antibody, we found that themajority of EpCAMþCD44þ cells identified using pan-CD44antibodies are CD44v8-10 cells. This is shown in a represen-tative sample GC101, where in the EpCAMþ fraction, 79% ofpan-CD44–expressing cells are CD44v8-10–positive (Fig. 3C;isotype controls are shown in Supplementary Fig. S2C). Thisobservation was further substantiated by analysis of two otherclinical samplesGC121 andGC123 showing thatmajority of theCD44 population in the tumor consists of CD44v8-10 (Fig. 3Dand E). Strikingly, CD44v8-10 is absent in hematopoieticCD45þ cells (Fig. 3C), in contrast to CD44s that is expressedin hematopoietic cells.

To determine that CD44v8-10 marks gastric CSCs, we pro-spectively isolated the CD44v8-10 cells and subjected themto cancer-initiation assay by limiting dilution analyses.CD44v8-10–specific antibody was used to fractionate

CD44v8-10þ cells from gastric tumor xenografts GC45 andGC84 to high purity (Fig. 4A) before performing limitingdilution assays. We injected the unsorted, CD44v8-10þ, andCD44v8-10� cell fractions into NSG mice at cell doses of 2,000,200, and 20. In the GC45 xenograft, the CD44v8-10þ fractionshowed a 51- (P ¼ 2.5 � 10�19) and 3.8-fold (P ¼ 0.00217)enrichment of CSCs compared with the CD44v8-10� andunsorted fractions, respectively (Fig. 4B). Similarly for GC84,CD44v8-10þ fractions were enriched for CSCs by 84- (P ¼1.4 �10�18) and 6-fold (P ¼ 0.00242) compared with CD44v8-10� and unsorted cell fractions, respectively (Table 2 andFig. 4B). We also serially transplanted CD44v8-10þ cells reiso-lated from CD44v8-10þ first transplant tumors of GC45 andGC84 into second transplant recipients, and observed thesame percentage of CD44v8-10þ and CD44v8-10� cells as thefirst transplant xenograft tumors (Fig. 4C). Histology of seriallytransplanted xenograft tumors recapitulated that of the initialxenografts (Fig. 4D), confirming that CD44v8-10 indeed marksa population of CSCs, which are able to form tumors as wellas regenerate the original tumor heterogeneity. However, as

Table 1. Limiting dilution assay for EpCAMþCD44þ cells

Gastric cancer cell source Cell dose Number of mice with tumors/number of mice injected (%)

GC16 Unsorted EpþCD44þ EpþCD44�

25,000 5/5 (100%) ND 3/5 (60%)10,000 4/5 (80%) ND 4/5 (80%)5,000 5/12 (42%) ND 3/5 (60%)2,000 2/9 (22%) ND 0/9 (0%)1,000 0/5 (0%) ND 0/5 (0%)500 0/3 (0%) 3/3 (100%) 0/3 (0%)100 0/8 (0%) 6/8 (75%) 0/8 (0%)20 0/8 (0%) 2/8 (25%) 0/8 (0%)

Estimated stem cell frequency(confidence intervals)

1 in 8,140 (4,782–13,855) 1 in 72 (35–148) 1 in 14,770 (7,586–28,758)

Estimated gastric CSC in 10,000 cells 1.2 138.9 0.68


5,000 2/2 (100%) 2/2 (100%) 2/2 (100%)4,000 3/3 (100%) 3/3 (100%) 3/3 (100%)2,000 12/12 (100%) 17/17 (100%) 7/12 (58%)500 3/5 (60%) 5/5 (100%) 5/15 (33%)400 8/10 (80%) 14/15 (93%) ND100 5/10 (50%) 13/15 (87%) 3/15 (20%)


1 in 267 (156–458) 1 in 83 (48–144) 1 in 1,455 (882–2,400)



2,000 12/13 (92%) 13/13 (100%) 10/13 (77%)500 4/7 (57%) 6/7 (86%) 2/7 (29%)300 5/6 (83%) 6/6 (100%) 2/6 (33%)50 2/13 (15%) 5/13 (38%) 2/13 (15%)


1 in 498 (282–880) 1 in 140 (76–259) 1 in 1,142 (660–1,977)


Abbreviation: ND, not determined.

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CD44v8-10� cells may consist of both CD44þ and CD44�

populations, we next compared the tumor-initiating potentialbetween CD44þ and CD44v8-10þ fractions and observed thatwhen injected at a dose of 200 cells, CD44v8-10þ cells formedtumors with higher frequency than CD44þ cells (Fig. 4E). Thisclearly demonstrates that CD44v8-10þ cells within the CD44þ

fraction have enhanced tumor-initiating capability comparedwith CD44þ cells.The sphere formation assay is widely used as in vitromethod

to evaluate stemcell activity in normal tissue aswell as putativeCSCs (25); hence, we established tumor spheres from explantsof GC38 xenograft tumor and confirmed that the sphere-forming cells are indeed human epithelial cells that expressEpCAM (Supplementary Fig. S3C and S3D). However, when weisolated CD44v8-10þ and CD44v8-10� cells from the spheroidculture, we found that both isolated subpopulations formedspheres in serum-free medium (Supplementary Fig. S3E andS3F). Moreover, other gastric xenograft lines did not survive aslong-term tumor sphere cultures regardless of CD44v8-10expression. As it has been noted that not all spheres originate

from stem cells (26), our results suggest that sphere-forming invitro limiting dilution assay may not be a reliable surrogate forthe more definitive in vivo limiting dilution assay in theidentification of surface markers for gastric CSCs.

CD44v8-10 is upregulated in gastric tumor samples andplays a functional role in tumor initiation

CD44 variant–specific primers were designed for quantita-tive real-time PCR (qRT-PCR) analysis on another cohort of 26gastric tumors and matched adjacent nontumor tissues. TheCD44v6 variant has also been reported to promote tumorgrowth and metastasis (27, 28); hence, we also investigatedits expression level in gastric tumors. Our results show thatCD44v8-10 expression was significantly elevated (P ¼ 0.0029)in tumor compared with adjacent nontumor tissue, whereasCD44s and CD44v6 expression levels were similar in bothtumor and adjacent nontumor tissue (Fig. 4F).

To determine whether CD44v8-10 plays a functional role ingastric cancer initiation, we next established and characterizeda clonal primary gastric cancer cell line GC38-adh from a

Figure 3. CD44v8-10 is the majorCD44 variant found on cells thatexpress surface CD44. A, PCR ofcDNA from xenograft lines GC16(lane 1), GC21 (lane 2), and GC38(lane 3). MW, molecular weightmarker. Diagram of human CD44gene structure showingalternatively spliced variant exonsv1-10 in colored boxes andconstant exons in gray boxes.CD44v8-10 comprises all constantexonsand variant exons 8–10whileCD44s contains only constantexons. PCR primers are located inconstant exons 5 and 16/17.B, qRT-PCR of CD44v8-10expression in xenograft tumors ofGC16, GC21, and GC38normalized to 18S rRNA internalcontrol. Error bars, mean � SD. C,flowcytometric analyses of surfaceexpression of CD44v8-10 inEpCAMþCD45� and CD45þ cellsisolated from tumor and adjacentnormal tissues of patient GC101.Proportion (%) of CD44v8-10–positive (CD44vþ) and CD44v8-10–negative cells within theEpCAMþCD45� or CD45þ

populations are shown in the topand bottom right quadrants,respectively. D, percentage ofCD44v8-10þ (CD44vþ) cells inEpCAMþCD45� population fromtumor and normal tissues of 3patients with gastric cancerGC101, GC121, and GC123.E, percentage of CD44v8-10þ cellswithin the CD44þ population ofpatients with gastric cancerGC101, GC121, and GC123.






patient with gastric cancer. We used GC38-adh cells for over-expression studies, as the percentage of CD44-expressing cellswas relatively low (5%) in this cell line. CD44s and CD44v8-10was stably overexpressed in GC38-adh cells (Fig. 5A) and theirproliferation rate was monitored over a period of 5 days,during which we observed that the in vitro growth rate ofthese cells was not significantly different (Fig. 5A). We nextsorted CD44s- and CD44v8-10–overexpressing cells andinjected these fractions into NSG mice at limiting cell dosesto determine their ability to initiate tumors. Our data show thatCD44v8-10–overexpressing cells were able to initiate tumorswith higher efficiency than vector control cells (P ¼ 0.0168;Supplementary Table S3A; Fig. 5B).

In reciprocal experiments, we performed shRNA-mediatedknockdown of total CD44 in gastric cancer cells. We usedTMK1 cells because >99.9% of the cells express the CD44v8-10isoform (Fig. 5C). We observed that injection of 200 CD44-

depleted cells (Supplementary Fig. S4B) did not form tumors,compared with scrambled control shRNA (Supplementary Fig.S4C). To "rescue" this knockdown phenotype, we expressedshRNA-refractory CD44v8-10 as well as CD44s and observedthat CD44v8-10 but not CD44s expression rescued the tumor-initiation potential of TMK1 cells in which total CD44 wasdepleted (Fig. 5D). As shown, the percentage of tumor-formingmice was higher when injected with CD44v8-10–rescued cellscompared with CD44s-rescued cells, and the tumors formedwere also larger (Supplementary Fig. S4D). In summary, thesefindings support the notion that CD44v8-10 plays an importantrole in tumor initiation.

DiscussionRecent developments in the stem cell field have further

validated theCSC concept. By using genetic techniques to tracethe cells during tumor progression in mice, three research

Figure 4. CD44v8-10 marks gastricCSCs. A, flow cytometric analysisof CD44v8-10þ and CD44v8-10�

(CD44vþ or CD44v�, respectively)cells in unsorted GC45 and GC84xenograft tumors and the purity ofsorted fractions. B, summary oflimiting dilution assays estimatingCSC frequency in unsorted,CD44vþ, and CD44v� cell fractionsfrom GC45 and GC84 xenografts.Differences between groups weretested for statistical significanceusingx2 test (degrees of freedom¼1; 95% confidence intervals).�, statistically significant P values.Detailed data for limiting dilutionanalysis and estimation of gastricCSC frequency are shown inTable 2. C, flowcytometric analysisof CD44vþ population in first andsecond serially transplantedxenograft tumors generated fromprimary CD44vþ cells. D, H&Estaining showing histology oftumors from first and secondxenograft transplants initiatedby CD44vþ cells. Scale bars,100 mm. E, comparison of tumor-initiating potential betweenCD44�,CD44þ, and CD44v8-10þ fractionsfrom xenograft GC119. Twohundred cells of the indicated cellfraction were injected andproportion of tumor-forming NSGmice is plotted. F, qPCR of CD44sand CD44v8-10 expression in 26primary tumor samples versusadjacent nontumor tissue,normalized to 18S rRNA internalcontrol. Horizontal bars, the meanvalue. Paired Student t test wasperformed for statisticalsignificance.

Lau et al.





groups have provided clear experimental evidence that CSCsexist and drive tumor growth (29–31), further emphasizing theneed to eliminate CSCs for effective eradication of cancer.We identified and enriched for gastric CSCs in primary

gastric cancer using EpCAM and CD44 cell surface markers,and further characterized these cells. Several other studieshave reported the use of CD44 as a gastric CSC marker.Takaishi and colleagues (6) have previously identifiedCD44þ cell populations from gastric cancer cell lines thatpossess features of CSCs, namely tumor sphere formation andtumor formation in vivo. Chen and colleagues (32) identifiedCSCs in a subpopulation of CD44þCD54þ cells from gastrictumors and peripheral blood of patients with gastric cancerbased on their capability to self-renew and differentiate in vitroand in vivo. However, three times the number of CD44þCD54�

and CD44�CD54þ cells could also form tumors in mice,suggesting that the combination of CD44 and CD54 may notbe sufficient to enrich forCSCs in gastric tumors. CD44þCD54þ

cells were more efficient in cancer initiation only when com-pared with total cells from biopsy samples, which consisted ofboth tumor and nontumor cells. More importantly, althoughCD44 marks gastric CSCs, it is also highly expressed inhematopoietic cells. Ideally, a targeted therapy should inhibitfunctions of CSCs but not normal cells. Hence, there is a need toidentify cancer-specific or CSCmarkers that are upregulated intumors.CD44 and its variants have been reported in gastric cancer

(33), and CD44 expression as determined by immunohis-tochemistry was found to correlate with poor prognosis inpatients with intestinal type gastric adenocarcinoma (34). Therelevance and significance of specific CD44 variants in cancerhave not been fully explored, and the nomenclature used todescribe these variants can be confusing. We isolated andidentified the transcripts of a predominant CD44 variantcontaining variable exons 8, 9, and 10 that was significantlyelevated in gastric tumors compared with normal gastric

tissue, referred to in this study as CD44v8-10 (Fig. 3A). TheCD44v8-10 surface protein can be recognized and identifiedusing a CD44v9-specific antibody (Supplementary Fig. S2A andS2B; refs. 20, 35), which allowed us to isolate and study CD44v8-10–expressing cells.

We used several patient-derived xenografts models in ouridentification of gastric CSCs and showed that pan-CD44 orCD44v8-10 expression alone was sufficient to predict enrich-ment of gastric CSCs. As CD44 is a knownWnt-signaling target(36), we characterized the xenograft models by microarrayanalyses and found that despite differences in expression ofWnt target genes, such as LGR5, ZNF43, ASCL2, CLDN2, SP5,and AXIN2 (36, 37), there was no correlation between geneexpression profiles of theWnt pathway and CD44v8-10 expres-sion (data not shown), indicating that molecular differences inthese xenografts had no bearing on the function of CD44v8-10in tumor initiation. We also evaluated p53mutation status in aselected number of xenograft lines because p53 has beenreported to inhibit expression of the CD44 cell surface mole-cule (38). Although we observed p53 mutations in somexenograft lines, we found no correlation between p53mutationstatus andproportion of CD44-expressing cells in the xenografttumor (Supplementary Table S4).

CD44v8-10 (CD44R1) was first described in colon cancer andwas found to be upregulated in primary andmetastatic tumorsbut rarely expressed in normal mucosa of adults (24). In theGan mouse, a genetic model for gastric tumorigenesis, themouse homolog of human CD44v8-10 is expressed in precan-cerous regions of the stomach, from which tumor-initiatingcells are thought to arise, suggesting that CD44v8-10 may be amarker for gastric tumor-initiating cells in the mouse (39).CD44v8-10 also seems to promote metastasis in colon, pan-creatic, and breast cancer cells (24, 40, 41), with significantpositive correlation to tumor recurrence and mortality (24).Using antibodies directed against the CD44 variant exon 9,CD44v9 expression in primary tumors was significantly and

Table 2. Limiting dilution assay for EpCAMþCD44v8-10þ (EpþCD44vþ) cells

Gastric cancer cell source Cell dose Number of mice with tumors/number of mice injected (%)

GC45 Unsorted EpþCD44vþ EpþCD44v�

2,000 13/13 (100%) 13/13 (100%) 8/13 (62%)200 13/19 (68%) 19/19 (100%) 3/19 (16%)20 4/10 (40%) 4/10 (40%) 0/10 (0%)


1 in 139 (82–237) 1 in 36 (17–78) 1 in 1,844 (988–3,440)

Estimated gastric CSC in 10,000 cells 72 277.8 5.4

GC84 Unsorted EpþCD44vþ EpþCD44v�

20,000 4/4 (100%) 4/4 (100%) 3/4 (75%)2,000 7/7 (100%) 7/7 (100%) 2/7 (29%)200 2/8 (25%) 7/8 (88%) 1/8 (13%)20 1/8 (13%) 3/8 (38%) 0/8 (0%)


1 in 454 (187–1,108) 1 in 75 (34–166) 1 in 8,859 (3,320–23,641)

Estimated gastric CSC in 10,000 cells 22 133.3 1.1






positively associated with tumor recurrence and mortality(35, 42). These studies indicate the importance of CD44v9 asa prognosticmarker but functional studies that defineCD44v9-expressing cells as gastric CSCs are lacking. Because theCD44v8-10 isoform includes exon 9, our current findings mayprovide further functional and mechanistic explanation forthese correlative observations.

Overexpression of CD44v8-10 but not CD44s resulted in cellswith higher tumor-initiating potential in vivo although we didnot observe any significant changes in proliferation uponectopic expression of either protein in vitro. This suggests thatCD44v8-10 may enhance tumor-initiation capability of gastriccancer cells by increasing their resilience to adverse conditionssuch as hypoxia or oxidative stress. Indeed, there is evidencethat CD44v8-10 stabilizes the cystine transporter xCT, thereby

promoting the ability of cancer cells to defend themselvesagainst reactive oxygen species (20). In light of these observa-tions, CD44v8-10þ cells may also be responsible for conferringresistance to therapeutic treatment; therefore, it is of para-mount importance to eradicate this subpopulation of cells foreffective treatment of gastric cancer.

In conclusion, we have shown that CD44v8-10 is the majorvariant found in CD44-expressing gastric cancer cells. TheCD44v8-10þ cell population is enriched with gastric CSCs, andCD44v8-10 plays a functional role in tumor initiation. Moreimportantly, CD44v8-10 expression is low in normal tissues(both epithelial and hematopoietic cells; Fig. 3C), thus, makingit an ideal target for directed therapy against gastric CSCs. Inaddition, our study has broader implications as CD44 alsomarks CSCs of numerous cancers (11), many of whichmay also

Figure 5. CD44v8-10 is upregulated in clinical gastric cancer and plays a functional role in tumor initiation. A, immunoblot of primary gastric cancer cellsGC38-adh stably expressing CD44s and CD44v8-10. Cell proliferation assay was performed over 5 days. Data points represent mean � SD of fivereplicates. Difference in slopes indicates cell proliferation rate. Results are representative of two independent experiments. B, xenograft tumor formationpotential of fractionated GC38-adh cells overexpressing CD44s and CD44v8-10. Chi-squared tests were performed for statistical significance (degreesof freedom ¼ 1). �, statistically significant P values. Data from two independent experiments are represented, with cell doses and number of miceused reflected in Supplementary Table S3A. C, top, CD44v8-10 expression in TMK1 cells analyzed by flow cytometry. Bottom, post-sort analysesof CD44-silenced TMK1 cells rescued with indicated shRNA-refractory genes. Percentage purity of sorted cells is indicated. These cells wereused in xenograft tumor formation assay. D, xenograft tumor formation in NSGmice by CD44-silenced TMK1 cells harboring the indicated shRNA-refractoryCD44 variants. Xenograft tumor formation potential for the indicated cell population was determined by limiting dilution assay. Differences betweengroups were tested for statistical significance using x

2 test (degrees of freedom ¼ 1; 95% confidence intervals). �, statistically significant P values.Detailed data for limiting dilution analysis are presented in Supplementary Table S3B.

Lau et al.





overexpress CD44v8-10. However, as pan-CD44 antibodieswere used for prospective isolation of CSCs in most studies,this remains to be investigated. CD44 plays diverse physiologicroles in many different tissues (43), thereby undermining itssuitability as a CSC target. For instance, injection of antago-nizing anti-CD44 antibody induces systemic shock inmice (44),and other side effects of anti-CD44 treatment have also beendocumented (45). Unlike CD44s that is expressed in manynormal tissues, CD44v8-10 is cancer specific; therefore, ourdiscovery of CD44v8-10 as a gastric CSCmarker opens a criticalwindow for therapeutic targeting of CSCs in other humancancers.

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

Authors' ContributionsConception and design: A. Shabbir, J.B.Y. So, S.L. ChanDevelopment of methodology: W.M. Lau, K.A.P. Lopez, A.Y.L. Tay, M. Salto-Tellez, J.B.Y. So, S.L. ChanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): W.M. Lau, H.S. Chong, K.A.P. Lopez, A.Y.L. Tay,M. Salto-Tellez, A. Shabbir, J.B.Y. So, S.L. Chan

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): W.M. Lau, J.B.Y. So, S.L. ChanWriting, review, and/or revision of the manuscript: W.M. Lau, A. Shabbir,J.B.Y. So, S.L. ChanAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): W.M. Lau, E. Teng, H.S. ChongStudy supervision: J.B.Y. So, S.L. Chan

AcknowledgmentsThe authors thank H. Saya, O. Nagano, and T. Masuko for providing the

CD44v8-10 antibody; M. Teh for assisting with histologic analysis of patientspecimens; T.L.M. Chia for technical assistance with preparation of histologysections and breeding of NSG mice; and M.G.K. Tan and V. Krishnan for criticalreview of this article.

Grant SupportThis research was supported by the National Research Foundation Singapore

and the Singapore Ministry of Education under the Research Centres of Excel-lence initiative and Singapore Stem Cell Consortium, A�Star Singapore (S.L.Chan).

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 12, 2013; revised February 18, 2014; accepted March 6, 2014;published OnlineFirst March 11, 2014.

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Lau et al.




Published OnlineFirst March 11, 2014.Cancer Res Wen Min Lau, Eileen Teng, Hui Shan Chong, et al. Stem CellsCD44v8-10 Is a Cancer-Specific Marker for Gastric Cancer

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