Characterization of Biomarkers of Tumorigenicand Chemoresistant Cancer Stem Cells inHuman Gastric CarcinomaPhu Hung Nguyen1,2, Julie Giraud1,2, Lucie Chambonnier1,2, Pierre Dubus2,3,4,Linda Wittkop2,5,6, Genevi�eve Belleann�ee4, Denis Collet4, Isabelle Soubeyran7,8,Serge Evrard2,7,8, Benoit Rousseau2,9, Nathalie Senant-Dugot2,10, Francis M�egraud1,2,4,Fr�ed�eric Mazurier11, and Christine Varon1,2
Abstract
Purpose:Gastric carcinomas are heterogeneous, and the currenttherapy remains essentially based on surgery with conventionalchemotherapy and radiotherapy. This study aimed to characterizebiomarkers allowing the detection of cancer stem cells (CSC)in human gastric carcinoma of different histologic types.
Experimental Design: The primary tumors from 37 patientswith intestinal- or diffuse-type noncardia gastric carcinoma werestudied, and patient-derived tumor xenograft (PDX) models inimmunodeficient mice were developed. The expressions of 10putative cell surface markers of CSCs, as well as aldehyde dehy-drogenase (ALDH) activity, were studied, and the tumorigenicproperties of cells were evaluated by in vitro tumorsphere assaysand in vivo xenografts by limiting dilution assays.
Results: We found that a subpopulation of gastric carcinomacells expressing EPCAM,CD133,CD166,CD44, and ahighALDH
activity presented the properties to generate new heterogeneoustumorspheres in vitro and tumors in vivo. CD44 and CD166 werecoexpressed, representing 6.1% to 37.5% of the cells; ALDHactivity was detected in 1.6% to 15.4% of the cells; and theALDHþ cells represented a core within the CD44þ/CD166þ
subpopulation that contained the highest frequency of tumori-genic CSCs in vivo. The ALDHþ cells possessed drug efflux prop-erties and weremore resistant to standard chemotherapy than theALDH– cells, a process that was partially reversed by verapamiltreatment.
Conclusions: CD44 and ALDH are the most specificbiomarkers to detect and isolate tumorigenic and chemore-sistant gastric CSCs in noncardia gastric carcinomas inde-pendently of the histologic classification of the tumor. ClinCancer Res; 1–12. �2016 AACR.
IntroductionGastric cancer is the fourth most common cancer in frequen-
cy and the third leading cause of cancer mortality in the world.Ninety-five percent of gastric cancers are gastric carcinomas,which are divided into two types depending on their localiza-tion in the stomach: adenocarcinomas of the cardia whoseetiology remains unclear, and noncardia gastric carcinomas forwhich the main factor is a chronic infection by Helicobacter
pylori (H. pylori). Infection with H. pylori, classified as a class 1carcinogen by the World Health Organization, induces a chron-ic inflammation evolving over decades from a chronic atrophicgastritis to intestinal metaplasia, dysplasia, and finally adeno-carcinoma (1, 2). Some cases also include Lynch syndromes(microsatellite instability, MSI) and Epstein–Barr Virus (EBV)infection. The classification of gastric carcinomas is basedessentially on histologic criteria. The Lauren classification dis-tinguishes two main subtypes, the intestinal type, which repre-sents the majority of the cases, and the diffuse type (3). Theintestinal type is composed of glands having more or lesspreserved their organization and differentiation state, or havingacquired intestinal characteristics; it is subclassified into tubu-lar, mucinous, or papillary carcinoma in the WHO classifica-tion of gastric carcinoma (4). The diffuse type is poorly cohe-sive, composed of isolated cells (often signet ring cells) pro-ducing mucins. These classification systems have little clinicalutility, as they cannot orientate patient therapy. With theexception of Her2 positivity which orientates toward a specifictreatment, treatment is still based on surgery combined withconventional chemotherapy and/or radiotherapy, and the5-year survival rates remain under 30% in most countries (5).
Recently, the Cancer Genome Atlas Research Network andWang and colleagues published a molecular profiling of gastriccarcinomas based on two studies with 295 cases and 100 cases,respectively. Both studies led to a classification of gastric carci-nomas into four main subtypes according to their molecular
1INSERM, U853 Helicobacter Infection, Inflammation and Cancer, Bordeaux,France. 2University of Bordeaux, Bordeaux, France. 3EA 2406, University ofBordeaux, Bordeaux, France. 4University Hospital Center of Bordeaux, Bor-deaux, France. 5INSERM, ISPED, Centre INSERM U1219 Bordeaux PopulationHealth, Bordeaux, France. 6Pole de Sant�e Publique, Service d'informationm�edicale, University Hospital Center of Bordeaux, Bordeaux, France. 7InstitutBergoni�e, Bordeaux, France. 8INSERM, U1012 Actions for onCogenesis under-standing and Target Identification in Oncology (ACTION), Bordeaux, France.9Service Commun des Animaleries, Animalerie A2, Bordeaux, France. 10SFRTransBioMed, Bordeaux, France. 11CNRS UMR 7292, GICC LNOx, Tours, France.
Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).
Corresponding Author: Christine Varon, University of Bordeaux, INSERM, U853Helicobacter Infection, Inflammation and Cancer, 146 rue Leo Saignat, BordeauxF33076, France. Phone: 33557579575; Fax: 33556796018; E-mail:[email protected]
profiles: (1) EBVþ tumors (frequent PIK3CA mutations, extremeDNA hypermethylation), (2) MSI tumors (elevated mutationrates, hypermethylation), (3) genomically stable tumors(enriched for the diffuse type; driver mutations include CDH1,RHOA, cytoskeleton, and cell junction regulators), and (4) chro-mosomal instability tumors (marked aneuploidy, focal amplifi-cation of tyrosine kinase receptors; refs. 6, 7). These studies wereperformed without distinguishing between cardia and noncardiagastric carcinomas, whose etiology is different.
Tumors are heterogeneous, composed of cells which are moreor less differentiated, and not all proliferative. Over the lastdecade, extensive research has focused on the discovery and thecharacterization of cancer stem cells (CSC) at the origin of cancersin numerous organs. Tumors are hierarchically organized withCSCs at the top of this pyramid and at the origin of tumorinitiation, heterogeneity, and propagation (8–10). The CSCscorrespond to a subpopulation of cells within the tumor definedby self-renewal, asymmetrical division, and differentiation pro-perties, giving rise to the more or less differentiated cells com-posing the tumor mass. CSCs can stay in quiescence undersome conditions, resist conventional therapies, and be at theorigin of tumor relapse and metastasis. The definition of CSCsremains largely operational and based on functional assays thatregister their self-renewal and tumorigenic properties, assessedby the formation of new heterogeneous tumors after xenograftin vivo, and of tumorspheres in particular culture conditionsin vitro (8–10).
Indeed CSC may display both genetic and phenotypic hetero-geneity, markers allowing their identification have been charac-terized in tumors of different organs, including CD133, CD44,and CD24 among those studied (8, 10–16). More recently, theactivity of aldehyde dehydrogenases (ALDH), intracellularenzymes involved in oxidation of aldehydes and retinoic acidsignaling, also led to the identification of CSCs in tumors of thebreast (17), lung (18), colon (19), and other organs (20).
In the stomach, the existence of CSCs has been subject todebate. The first study performed by Takaishi and colleagues on
gastric carcinoma cell lines proposed CD44 as a gastric CSCmarker, but this marker was expressed in three out of six celllines studied, and confirmation in primary tumors was lacking(21). Then, the study performed by Rocco and colleagues on12 human primary gastric carcinomas failed to demonstratetumor-initiating properties of CD133þ- and CD44þ-sorted cellsafter xenograft in both NOD/SCID and nude immunodeficientmice (22).
Another important point concerns the origin of the CSCs. AsHoughton and colleagues and our group reported in mousemodels of Helicobacter-induced gastric carcinogenesis, gastricdysplasia and carcinoma may originate from the transformationof a local epithelial stem cell or of a bone marrow (BM)–derivedstem cell (23–25). In this model, dysplastic lesions were com-posed of CD44þ cells, regardless of their BM or local origin (24).In addition, the heterogeneity of gastric carcinomas suggests thatgastric CSCmarkers, if they indeed exist, may be different accord-ing to the origin and/or the histologic type of gastric carcinoma.
In this study, we performed an extensive screening of theexpression of putative cell surface markers of CSCs as well asALDH activity in order to identify biomarkers allowing thedetection and isolation of tumorigenic and chemoresistant CSCsin human primary intestinal- and diffuse-type noncardia gastriccarcinoma.
Materials and MethodsHuman samples and mouse xenografts
Fresh tumors samples were collected from gastric surgicalwastes from patients who underwent gastrectomy for noncardiagastric carcinoma and for whose informed consent wasobtained. Fresh samples of tumor and paired nontumor tissueswere transported in DMEM medium with 20% FCS, 50 IU/mLpenicillin, 50 mg/mL streptomycin, 50 mg/mL vancomycin, and15 mg/mL amphotericin-B. Samples were minced in smallpieces of 2 mm � 2 mm size and were subcutaneously trans-planted into the right dorsal flank of 7-week-oldmale NSGmiceunder 2.5% isoflurane anesthesia (Belamont). Alternatively,after mechanical mincing, cells were dissociated by incubationin a solution of 1 mg/mL collagenase IV and 0.2 mg/mLhyaluronidase in DMEM (Sigma) for 1 hour at 37�C withshaking (15), then suspended in 100 mL of 7 mg/mL ice-coldMatrigel (BD Biosciences) for subcutaneous injection. Xeno-grafts were carried out within 3 to 5 hours following gastrec-tomy. The tumor size was monitored with callipers once a week,and tumor volume was estimated as (D2 x d)/2, where D is thelarge diameter and d is the small diameter (26). At the end ofthe experiments (until 10 months after engraftment for theprimary xenograft) and when tumor reached approximately500 mm3, mice were sacrificed by cervical dislocation andtumors were immediately harvested and processed for analyses.Secondary tumors were amplified subcutaneously in miceby serial transplantation of pieces of tumor bulk, or by injectionof tumor cells in Matrigel. For xenograft experiments in extremelimiting dilution assay (ELDA), 10,000 to 30 FACS-sorted cellswere subcutaneously injected with Matrigel; tumor size wasrecorded twice a week.
Gastric carcinoma cell linesGastric carcinoma cell lines were cultured in DMEM/F12
media for AGS (ATCC CRL1739) and in RPMI1640 media
Translational Relevance
We report the screening of the expression of 10 cell surfacemarkers and aldehyde dehydrogenase (ALDH) activity on cellsfrom primary gastric carcinoma. We found that a subpopula-tion of tumor cells expressing CD133, CD166, CD44, andALDH presented cancer stem cell (CSC) tumorigenic proper-ties in vitro and in vivo. Among them, ALDHþ cells represented1.6% to 15.4% of the tumor cells and contained the highestfrequency of tumorigenic CSCs before CD44þ cells. In addi-tion, the tumorigenic CD44þALDHþ cells possessed drugefflux and chemoresistance properties, constituting the cellsto target in the development of new therapy. Results alsoshowed that CD44, which is poorly expressed or absent inhealthy gastric epithelium, is overexpressed in gastric carcino-ma and may constitute a good biomarker for the detection ofCSCs by standard immunohistochemistry on patient tissuesamples, whereas detection of CSCs possessing a high ALDHactivity is not yet possible by standard immunohistochemistryand involves many ALDH isozymes.
for NCI87 (ATCC CRL-5822), MKN45, MKN74, MKN7, andMKN28 (all from RIKEN) cells, supplemented with 10% heat-inactivated FCS, 50 IU/mL penicillin and 50 mg/mL strepto-mycin (all from Invitrogen) at 37�C in a 5% CO2 atmosphere(26, 27). All cell lines were routinely verified mycoplasmafree (by PCR) and were tested and authenticated by shorttandem repeat (STR) profiling within 6 months preceding theexperiments (last STR profiling report, October 2015; LGCStandards). Cell viability was assessed by the trypan blueexclusion method.
Flow cytometry analysisCells were dissociated by collagenase/hyaluronidase proce-
dure from fresh patient-derived tumor xenografts (PDX),passed through a 70-mm mesh filter (BD), and red blood cellswere removed by incubating in a solution containing 2 mmol/LKHCO3, 0.1 mmol/L EDTA, and 170 mmol/L NH4Cl for 8minutes at 4�C. Then, 100,000 cells in 100 mL PBS-0.5% BSA-2mmol/L EDTA (Sigma) were stained with 3 to 5 mL of fluores-cent-labeled primary antibodies including EPCAM-FITC (StemCell Technologies) or EPCAM-VioBlue (MACS-Miltenyi Bio-tec), CD10-PE, CD24-PE, CD73-PE, CD49f-PE, CD105-PE,CD166-PE, CD90-PECy5, CD44-PE, CD44-APC, CD338-APC(all from BD), and CD133-PE (MACS-Miltenyi Biotec) for 20minutes at 4�C. Cells were rinsed twice with PBS-0.5% BSA-2mmol/L EDTA containing 50 mg/mL 7-aminoactinomycin-D(7-AAD; BD) before being analyzed using a FACSCanto IIinstrument and DIVA software (BD; refs. 26, 27). The ALDE-FLUOR Kit (Stem Cell Technologies) was used to detect ALDHactivity according to the manufacturer's instructions. Dead cellswere excluded based on light scatter characteristics and 7-AADpositivity. For SP cells analysis, cells were incubated with 10 mg/mL Hoechst-33342 in HBSS-2% FCS for 60 minutes at 37�C or,when indicated, in ALDEFLUOR buffer for 30 minutes at roomtemperature, with or without 100 mmol/L verapamil or 50mmol/L reserpine (Sigma), and then washed with ice-coldHBSS-2% FCS. The Hoechst-33342 dye was excited at 375 nm,and its fluorescence was dual wavelength analyzed (blue, 402–446 nm; red, 650–670 nm; ref. 28). Cell sorting was performedon 5 to 10 million cells stained with primary fluorescent-labeled antibodies or ALDEFLUOR reagent and 7-AAD, on7-AAD–negative cells using a FACSAria (BD).
Tumorsphere assayA total of 1,000 FACS-sorted cells were plated in nonadher-
ent 24-well plates (or alternatively 200 cells in 96-well plates)previously coated with a 10% poly(2-hydroxyethyl methacry-late) solution in 95% ethanol (v/v; Sigma), in DMEM-F12media supplemented with 20 ng/mL human-epidermal growthfactor, 20 ng/mL basic-fibroblast growth factor, 5 mg/mL insu-lin, 0.3% glucose, 50 IU/mL penicillin, and 50 mg/mL strepto-mycin (Sigma; ref. 26). For PDX cells, the media were supple-mented with 5% FCS for the first 2 days of culture and werethen replaced by serum-free media. After 7 days, the number ofspheroids/well was counted under light microscopy using a�20 objective. For drug treatment experiments, 5-day tumor-spheres grown in nonadherent 96-well plates (8 < n < 10 percondition) were treated with 10 to 20 mmol/L of verapamil with5-fluorouracil, doxorubicin, and cisplatin (all from Sigma).After 48 hours, the number of tumorspheres was recorded. Theself-renewal ability of residual viable cells dissociated from
tumorsphere by trypsin/EDTA procedure and seeded in newnonadherent 96-well plates (8 < n < 10 per condition) wasevaluated after 6 to 9 days.
Ethic statements, histology, immunohistochemistry, immuno-fluorescence procedures, and statistical analysis are described inSupplementary Materials.
ResultsEstablishment of a mouse PDX model of diffuse- andintestinal-type noncardia gastric carcinoma
Fresh gastric carcinoma and nontumor tissue samples werecollected by pathologists upon surgical resection from consentingpatients who underwent gastrectomy for noncardia gastric carci-noma at the University Hospital Center and the Bergoni�e Institutein Bordeaux. Among the 37 cases studied, the median age was 72years, 59.5% were males and 54.1% did not receive preoperativechemotherapy (Supplementary Table S1). Approximately half ofthe tumors were tubular and one third were poorly cohesiveaccording to the WHO and Lauren classifications of gastric car-cinomas. More than half were high grade (51.4%), highly pen-etrant (T4¼ 51.3%) with lymph node invasion (N1–4¼ 67.6%),and at stages 3 to4 (51.4%).When tissue sizewas sufficient, pieceswere dissociated by enzymatic procedures, and single cells wereanalyzed by flow cytometry for the expression of EPCAM, anepithelial marker, in combination with CD24, CD133, and CD44as putative CSC markers, and 7-AAD to exclude dead cells(Fig. 1A). To overcome the problem of the small size of thehuman samples which was a limiting factor in the study, wedeveloped PDX models in NSG immunodeficient mice. For this,small pieces of fresh tissue from the patient's biopsy were sub-cutaneously xenografted in mice (3 < n < 10 mice per case).Nontumor tissues xenografted as controls never led to tumorgrowth over the 10months. Among the 37 tumors xenografted, 8led to the growth of secondary tumors serially transplantable inmice; 7 were intestinal type (GC04, GC07, GC10, GC35, GC40,and GC44), and 1 was diffuse type (GC06; Supplementary TableS1). There was no significant association between PDX growthand the following characteristics: patient's gender, age, preoper-ative treatment, WHO and Lauren classifications of gastric tumorhistologic type, grade, tumor-node-metastasis classification, andstage (Supplementary Table S1). PDXs reached a 500 mm3 sizebetween 2 to 6 months (mean 16.7 � 3.4 weeks) after the firstpassage (P) (P1) in mice, and earlier following successivepassages (after 10.9 � 5.9 weeks at P2 and 7.2 � 0.8 weeks atP5; Fig. 1B). Case GC42, a mucinous type according to theWHO classification, was excluded because tumors developedslowly and were mostly composed of mucus, rendering its studyimpossible. Histopathologic analyses confirmed that the PDXsobtained between P1 and P5 remained similar to the respectivepatients' primary tumor for all cases (Fig. 1C) except GC07, whichappeared to dedifferentiate after P2 and therefore was excluded(data not shown).
Evaluation of CD24, CD133, and CD44 cell surface markerexpression on patients' gastric tissues and PDXs
The expression of CD24, CD133, and CD44 was evaluated byflow cytometry in live (7-AAD negative) EPCAMþ epithelialcells dissociated from freshly collected paired nontumor andtumor gastric tissue samples from 7 cases. In nontumor gastrictissues, CD24 and CD133 were expressed in about half of the
Biomarkers of Tumorigenic and Chemoresistant Gastric CSCs
cells, whereas CD44 was expressed only in 10% � 9% ofcells. In paired tumors, CD24, CD133, and CD44 expressionwas significantly higher compared with nontumor cells, CD24being expressed in most of the tumor cells (90 � 8%), CD133in 71% � 17%, and CD44 in 27% � 17% of the tumor cells(Fig. 2A). The percentage of tumor cells expressing these mar-kers were then evaluated on cells dissociated from freshlycollected serial xenografts from P1 to P4 of the 6 PDXs cases(Fig. 2B). The percentage of cells expressing the differentmarkers remained stable in serial PDXs from P1 to P4, EPCAMbeing expressed in most of the cells (>80%) followed by CD24and CD133 expressed at a relatively high proportion of cells,except in GC35. In all cases, CD44 was expressed in less than athird of total cells. These results were confirmed by immuno-histochemistry analyses of the expression of CD44 on patient'snontumor and tumor tissues and on the corresponding P2–3PDXs for the 6 cases studied (Fig. 2C). In the nontumor area,CD44 was expressed at a low level, preferentially in cells in theisthmus region of gastric glands in the area of gastritis, as
previously reported (26). In patients' tumors, CD44 wasexpressed in some tumor cells but not all, mainly at theperiphery of the tumor islets for the intestinal-type tumors.A similar pattern of CD44 expression was observed in PDXs,confirming that the cellular heterogeneity of the primary tumorwas reproduced in serial PDXs.
We then analyzed the expression of seven additional cellsurface markers as putative markers of gastric CSCs, i.e., CD10,CD49f, and CD166 described in CSCs of other organs (29–31),CD73, CD90, and CD105 as the main markers of mesenchymalstem cells (27, 32), as well as ALDH activity in cells from fivePDXs (freshly collected at P2–P4) and five gastric carcinomacell lines (seven intestinal type: GC04, GC10, GC35, GC44,MKN74, MKN7, NCI87; and three diffuse type: GC06, AGS,MKN45; Fig. 3A). CD10 was negative except for 2 of the 10cases studied. CD49f was expressed in more than 80% of the
Establishment of mouse xenograft models using primary tumors from patients with noncardia gastric carcinomas (GC). A, schematic representation ofthe strategy used to collect, analyze, and perform serial tumor xenografts in NSG immunodeficient mice from primary noncardia gastric carcinomafreshly collected from patients who underwent gastrectomy. At each passage (P) in mice, histology and flow cytometry analyses were performedrespectively on tissue samples and on cells freshly dissociated by enzymatic procedures. In vitro tumorsphere assays and in vivo CSC frequencydetermination were monitored after cell sorting by FACS of cell subpopulations based on the expression of EPCAM, CD133, CD44, and ALDH activityfollowing the second passage in mice (P2). B, number of weeks when a tumor size reached 500 mm3 after serial transplantation in mice (from P1 to P5).Histograms represent the mean � SD for each case, and numbers represent the global mean � SD of all cases (1 < n < 12). C, representative images ofhematoxylin–eosin saffron staining of primary tumors from patients and corresponding P2–P3 tumor xenografts. Scale bars, 50 mm.
cells in all cases, similar to EPCAM. For the other markers, theexpression pattern was more homogeneous in PDXs than in celllines which exhibited more heterogeneity, being either highlypositive or negative. CD133 was expressed only in PDXs cellsand not in gastric carcinoma cell lines. In PDXs, CD49f andCD24 were highly expressed, followed by CD133 and CD90expressed in nearly half of the cells, then by CD73 expressed inmore than a third of the cells. At a level similar to CD44, CD166was expressed in 21% � 13% of the cells. CD105 expressionand ALDH activity were detected in only 9% � 6% and 8% �5% of the cells, respectively (Fig. 3A). Results from flowcytometry costaining analyses revealed that CD166 and CD44were coexpressed and detected the same cell subpopulation(Fig. 3B). The majority of CD44þ cells were positive for CD24,CD133, and CD73. Less than 50% of CD44þ cells were positivefor ALDH, CD105, and CD90. Interestingly, ALDH activity butnot CD90 and CD105 expression was recorded mainly inCD44þ cells, showing that ALDHþ cells representing a corewithin the CD44þ subpopulation of cells (Fig. 3B).
In order to evaluate the tumorigenic properties of the cellsexpressing or not CD44, CD133, CD73, CD166, CD90, andCD105, P2–P3 tumors of 3 PDX cases, GC04, GC06, and GC10,were freshly dissociated and 7-AAD-EPCAMþ cells either positive
or negative for these markers were sorted by FACS and submittedto the tumorsphere assay. Similar experiments were performed on7-AAD–FACS-sorted cells based on ALDH activity. In all of thecases, EPCAMþCD133þ, EPCAMþCD44þ, EPCAMþCD73þ,EPCAMþCD166þ, and ALDHþ cells formed significantly moretumorspheres after 10 days of in vitro culture than their respectivenegative counterparts (Fig. 3C). CSCs forming tumorspheres wereessentially present in CD44þ, CD166þ, and ALDHþ subpopula-tions, and to a lesser extent in CD133þ and CD73þ subpopula-tions; they were essentially CD90� and CD105�. The high tumor-sphere capacity of ALDHþ cells was confirmed on both MKN45and MKN74 cell lines (Fig. 3C).
To confirm these results in vivo, xenografts were performed inmice with 7-AAD-EPCAMþ FACS-sorted cells based on theexpression of CD133 and CD44 on GC04, GC06, and GC10PDXs. The same experiments were performed based on ALDHactivity on GC06, GC10, MKN45, and MKN74 cells (Table 1). Inall cases, tumors developed at a significant higher frequency inEPCAMþCD44þ cells (1/29 to 1/1,020) than in their respectiveEPCAMþCD44� cells (1/568 to 1/28,963), and in EPCAMþ
CD133þ cells (1/105 to 1/1,911) than in their respectiveEPCAMþCD133� cells (1/781 to 1/66,876; Table 1). CSC fre-quency was higher in EPCAMþCD44þ cells than in EPCAMþ
Expression of CD24, CD133, and CD44 on gastric epithelial cells from patients and gastric tumor xenografts. The percentage of cells expressing CD24,CD133, and CD44 was determined by flow cytometry analyses on cells dissociated from freshly collected specimens. A, analyses were performed on7-AAD-EPCAMþ gastric epithelial cells dissociated from paired nontumor (healthy) and tumor gastric mucosa samples from patients (n ¼ 7). Bars, median.� , P < 0.05. B, histograms represent the cumulated percentages of positive cells determined for each marker in serial xenografts. Note: CD24 andCD44 expressions were not evaluated on GC06 P1. n, number of tumors analyzed per passage and per case. P1, 1 < n < 3; P2 to P4, 3 < n < 17. C, representativeimages of CD44 detection by immunohistochemistry on paired tumor and nontumor distant mucosa from patients and corresponding P2–P3 tumorxenografts in mice. Scale bars, 50 mm.
Biomarkers of Tumorigenic and Chemoresistant Gastric CSCs
CD133þ cells, confirming that CD44 is a better marker of gastricCSCs than CD133, as also determined by the in vitro tumor-sphere assays. ALDHþ cells led to the development of tumors ata significant higher frequency than the respective ALDH� cells(1/38 to 1/746 for ALDHþ cells vs. 1/372 to 1/8,024 for ALDH�
cells) for all cases studied, with the exception of MKN45 whichwas highly tumorigenic at the doses studied (Table 1). For bothPDXs cases GC10 (intestinal type) and GC06 (diffuse type), theCSC frequencies were high in ALDHþ cells (1/81 and 1/38,respectively) and in EPCAMþCD44þ cells (1/29 and 1/352,respectively), and both were higher than in EPCAMþCD133þ
cells (1/105 and 1/1,658, respectively), suggesting that ALDHand CD44 are more specific markers of gastric CSCs thanCD133. Finally, we showed that the CSCs contained inEPCAMþCD44þ and ALDHþ FACS-sorted cells generatedtumors that recapitulated the phenotypic heterogeneity of theinitial tumors, giving rise both to CD44þ cells containing CSCsand to more differentiated CD44� cells (Fig. 4A). The asym-metric division and differentiation properties of these CSCs wasconfirmed in vitro with MKN45 cells; ALDHþ FACS-sorted cells
were able to reproduce heterogeneous tumorspheres in vitro,composed of a similar proportion of cells with ALDHþ activitycompared with the initial situation and expressing CD44, and ofCD44þALDH� and CD44�ALDH� cells incorporating theHoechst-33342 stain (Fig. 4B). In addition, the CD44þALDHþ
FACS-sorted cells generated more tumorspheres than the CD44þ
ALDH� cells which generated less but still a significant numberof tumorspheres and which therefore could correspond toprogenitor/transit amplifying cells. The CD44�ALDH� cells maycorrespond to more differentiated cells with very limited pro-liferation capacities as they formed significantly less or notumorspheres compared with the CD44þALDH� and CD44þ
ALDHþ cells (Fig. 4C).
ALDHþCSCs aremore resistant to conventional chemotherapythan ALDH– cells in gastric carcinoma
The combined analysis of CD44 expression, ALDHactivity, andHoechst-33342 incorporation was assessed on live MKN45tumorsphere (as in Fig. 4B) during their development. In young,small tumorspheres (after 5 days), most cells were positive
Table 1. Gastric cancer–initiating cell frequencies determined on FACS-sorted cells according to the expression of CD133, CD44, and ALDH after tumor xenografts inlimiting dilutions in NSG mice
Number of tumors/number of transplanted miceNumber of transplanted cells
NOTE: Case 1, GC04; case 2, GC06; case 3, GC10; case 4, MKN45; case 5, MKN74.aTherewas complete data separation, thus estimatesmaynot be reliable. Furthermore, the single-hit assumption that one cell is sufficient for a positive responsemaynot be true.
Figure 3.Expression of putative markers of CSCs and tumorigenic properties of corresponding cell subpopulations of PDXs and gastric carcinoma (GC) cell lines. A,the percentages of cells expressing EPCAM, CD24, CD133, CD44, CD10, CD49f, CD73, CD166, CD90, CD105, and ALDH activity were determined by flowcytometry analyses on 7-AAD cells from 5 PDXs after P2–P4 (PTs: GC04, GC06, GC10, GC35, and GC44) and 5 gastric carcinoma cell lines (CLs: AGS,MKN28, MKN45, MKN74, and NCI87) of diffuse and intestinal types. Bars, median. B, representative dot-plot analyses of CD24, CD133, CD73, CD166, CD90,and CD105 stained with PE and PECy5-labeled antibodies and ALDH activity determined by ALDEFLUOR assay in combination with anti-CD44/APCantibodies on GC10 cells (top). Quantification of the percentage of positive cells for the different markers in the PDXs (5 < n < 6). Bars, median. C,7-AAD tumor cells were sorted by FACS based on the expression of the indicated markers and on ALDH activity; and the positive and negativesubpopulations of sorted cells were submitted to in vitro tumorsphere assays. Representative images of cell sorting by FACS and phase contrast microscopyof tumorspheres formed by GC10 cells after 10 days of culture in nonadherent conditions in vitro (top). Quantification of the number of tumorspheresformed by FACS-sorted cells after 5 to 10 days of culture (bottom). Results represent the mean � SD (n ¼ 8 per condition). � , P < 0.05 for all cases studied.#, P < 0.05 for only 2 of 3 cases studied. Scale bars, 50 mm.
Biomarkers of Tumorigenic and Chemoresistant Gastric CSCs
for both CD44 and ALDH activity and were negative for Hoechst-33342 (Supplementary Fig. S1). When tumorspheres becamebigger (after 10–15 days), only a fraction of cells remainedCD44þALDHþ and Hoechst-33342, which may correspond toCSCs, with the appearance of CD44þALDH� Hoechst-33342þ
cells which may correspond to progenitor/transit amplifyingcells (Supplementary Fig. S1).
An important property of CSCs is to be resistant to conven-tional therapies, leading to tumor recurrence and metastasis aftertreatment (8, 9). Verapamil treatment, known to inhibit drugefflux systems, restored Hoechst-33342 incorporation in ALDHþ
cells in MKN45 and GC10 tumorspheres in vitro (Fig. 5A). Thiseffect was confirmed by flow cytometry analyses on MKN45cells and confirmed at a lesser extent with reserpine, anotherinhibitor of drug efflux systems (Fig. 5B). The drug efflux prop-erties of CSCs are usually assessed by the functional analysis of theSide Population (SP), a minor subpopulation of cells defined by
Hoechst-33342 stain efflux properties in specific experimentalconditions. In these particular experimental conditions, we founda consistent proportion of Hoechst-SP cells in MKN45 cell line(3.9% � 0.8 % of total cells; Fig. 5C) but not in MKN74 cell line(<0.2%, data not shown) as previously reported by others (28).Thepercentage ofMKN45SP cellswas significantly decreasedwithverapamil and reserpine compared with untreated cells (0.6% �0.1% and 1.2% � 0.2%, respectively, vs. 3.9% � 0.8%; Fig. 5C).The determination of ALDH activity within SP cells was thenassessed in the experimental conditions of theALDEFLUORassay,those of the SP cells assay being incompatible and leading toALDEFLUOR substrate clearing (Supplementary Fig. S2; Supple-mentary Methods). Results showed that most of the ALDHþ
cells were present in the Hoechst-subpopulation of SP-like cells(Fig. 5D). Combined together, these results suggest that ALDHþ
cells could possess drug efflux properties and may be moreresistant to treatments.
CD44þ and ALDHþ cells generate heterogeneous tumors in vivo and tumorspheres in vitro. A, representative images of CD44 detection byimmunohistochemistry on tumors obtained after xenograft of EPCAMþCD44þ and ALDHþ FACS-sorted cells of the indicated PDXs and gastriccarcinoma (GC) cell lines. Scale bars, 50 mm. B, MKN45 ALDHþ FACS-sorted cells were submitted to the tumorsphere assay for 8 days, and then analyzedfor CD44 expression and ALDH activity. Representative images of fluorescent imaging of CD44 stained with anti-CD44/PE antibodies (in red), ALDHactivity detected by ALDEFLUOR reagent (in green), and nuclei staining with Hoechst-33342 (in blue), and of flow cytometry analysis (right) ofCD44 stained with anti-CD44/APC antibodies and ALDH activity detected by ALDEFLUOR reagent. Scale bar, 25 mm. C, cells from MKN45, NCI-87,and GC07 PDX were stained as in B and with 7-AAD (to exclude 7-AADþ dead cells) and anti-EPCAM/VioBlue antibodies for GC07 cells dissociatedfrom a fresh PDX (to select EPCAMþ carcinoma cells). The 7-AAD-(EPCAMþ) cells were sorted by FACS on the expression of CD44 and ALDHactivity and submitted to the tumorsphere assay. Plots (min to max) represent the number of tumorspheres formed per 200 cells seeded per well after5 to 8 days of culture (n ¼ 10 per condition). � , P < 0.05. B and C, numbers indicate the percentage of cells in each quarter.
To explore this hypothesis, first, the sensitivity of MKN45 andMKN74 cells to drugs commonly used in gastric carcinomatreatment was determined on cell viability in adherent cultureconditions. Both cell lines showed dose-dependent antiproli-ferative responses to 5-fluorouracil, doxorubicin, and cisplatintreatments, which were increased in the presence of verapamil(Supplementary Fig. S3). Second, MKN45 and MKN74 cellswere sorted by FACS based on ALDH activity, and the viabilityof ALDHþ and ALDH� cells was evaluated in the presence of5-fluorouracil, doxorubicin, and cisplatin in combination withverapamil in vitro. This assay cannot be performed on cells from
PDXs, given that these cells are not cultivable in adherent cul-ture conditions and that only ALDHþ cells and not ALDH� cellscan form tumorspheres in vitro (Fig. 3C). In both cell lines,ALDHþ cells were more resistant than ALDH� cells to both5-fluorouracil and doxorubicin treatments but not to cisplatinat the dose studied (Fig. 5E). Verapamil treatment sensitizedALDHþ cells to these chemotherapies (Fig. 5E). This effect wasconfirmed on the formation of tumorsphere by both cell lines,in which verapamil treatment potentiated significantly thereduction of tumorsphere number in response to 5-fluorouracil,doxorubicin, and cisplatin treatments (Fig. 5F). The same result
Figure 5.
ALDHþ cells have drug efflux properties, and verapamil treatment sensitizes them to chemotherapeutic drugs. A, representative images offluorescent imaging of CD44 stained with anti–CD44-PE antibodies (in red), ALDH activity detected by ALDEFLUOR reagent (in green), and nucleistaining with Hoechst-33342 (in blue) on 12-day MKN45 tumorspheres and on 5-day GC10 tumorspheres. Tumorspheres were treated or not (control)with verapamil for 10 minutes before staining. Dotted circles point out ALDHþ cells without or with Hoechst-33342 stained nuclei. Bars, 10 mm. B,histogram represents the percentage of Hoechst-33342 cells analyzed by flow cytometry on cells dissociated from tumorspheres (A) treated or notwith verapamil 100 mmol/L and reserpine 50 mmol/L (n ¼ 3). C, analysis of the SP and main population (MP) in MKN45 cells treated or not with verapamil100 mmol/L and reserpine 50 mmol/L and incubated with Hoechst-33342 in HBSS-2% FCS for 60 minutes (n ¼ 3). D, dot-plot analyses of ALDH activityin Hoechst– (SP-like) and Hoechstþ (MP-like) cells, detected after 30-minute incubation in ALDEFLUOR buffer at 37�C, then 30-minute incubation withHoechst-33342 at room temperature (n ¼ 3). E, percentage of viable ALDHþ (black bars) and ALDH� (white bars) FACS-sorted MKN45 and MKN74 cells after48 hours of adherent culture and treatment without (control, CT) or with 10 mmol/L verapamil with or without 50 mmol/L 5-fluorouracil (5-FU), 1 mmol/Ldoxorubicin (DOXO), or 50 mmol/L cisplatin. B–E, results represent the mean � SD. F and G, plots (min to max) represent the number of MKN45 andMKN74 tumorspheres formed: F, after a 48-hour treatment of 5-day tumorspheres without (control, CT) or with verapamil (dotted bars), 5-FU, DOXO,and cisplatin as in E; G, after 5 days by cells dissociated from residual-treated tumorspheres (from experiment described in F). E–G, 8 < n < 10 per condition.� , P < 0.05.
Biomarkers of Tumorigenic and Chemoresistant Gastric CSCs
was obtained on tumorspheres of GC10 PDX (Supple-mentary Fig. S4). Self-renewal assays with residual cells fromMKN45- and MKN74-treated tumorspheres confirmed thatthe combination of verapamil with these conventional chemo-therapeutic drugs significantly reduced the number of tumori-genic CSCs (Fig. 5G). Altogether, these results indicate that,within the CD44þ subpopulation of tumor cells, the determi-nation of ALDH activity allows the detection and isolation ofCSCs with tumorigenic and chemoresistant properties in gastriccarcinoma.
DiscussionIn this study, we characterized the expression of cell surface
biomarkers and ALDH activity of gastric CSCs in intestinal- anddiffuse-type noncardia gastric carcinomas.
Contrary to the situation in colon cancer in which CD133þ
cells containing colon CSCs represent a small and rare sub-population within tumors (13, 14), we demonstrated thatCD133þ cells (detected by similar experimental procedures)were frequent in gastric carcinoma. We also showed that CD133was a less specific marker for the enrichment of gastric CSCsthan CD44 and ALDH, as demonstrated by their lower capacityto form tumorspheres in vitro and a new tumor after xenograftin vivo. CD44 was expressed in all patient-derived primarygastric carcinomas but not in the healthy gastric mucosa or ata very low level in the isthmus of the corpus gastric glandswhere stem cells reside. We and others previously reported thatthese CD44þ stem/progenitor cells expand from the isthmustoward the base of the unit in metaplastic and dysplastic areasinduced in response to chronic H. pylori infection (24, 26, 33).This occurs via an epithelial–mesenchymal-like transition, con-ferring CSC-like properties to CD44þ cells (25, 26). CD44expression has been reported in gastric carcinoma (34–37).A recent study demonstrated that CD44 inhibition by peptideinhibitors prevented the development of cellular hyperproli-feration and chronic atrophic gastritis in animal models ofH. pylori–induced gastric carcinogenesis (33, 38).
Interestingly, we showed that CD166 was coexpressed withCD44, and, as a consequence, CD166þ cells presented the sametumorigenic properties as CD44þ cells in vitro. Similar analyses ofin vitro coexpressionwith CD44 and tumorigenic properties led tothe conclusion that the gastric CSC phenotype corresponds toEPCAMþ, CD24þ, CD133þ, CD73þ, CD90�, CD105�, CD166þ,and CD44þ, associated with ALDH activity. Finally, in all PDXsstudied, cells with ALDH activity represented the smallest sub-population of cells compared with all othermarkers studied, withhigh tumorigenic properties both in vitro and in vivo, and withasymmetric division and differentiation properties reproducingthe heterogeneity of the initial tumors. As for other cancers, wemust consider that the gastric CSC phenotype may be plastic,subjected to regulation by the surrounding tumor microenviron-ment in vivo. Recent work has demonstrated that breast CSCscoexist between two different phenotypic states: a more quiescentand invasive, mesenchymal-like state characterized by a CD24�
CD44þ phenotype and located mainly at the tumor peripheryand invasive front, and a more proliferative epithelial-likestate, characterized by ALDH activity and located more centrally(9, 39). However, unlike the situation in breast cancer, we haveshown that gastric CSCs express both CD44 and ALDH activity,and that ALDH activity reveals a subpopulation within the
CD44þ cells (see Fig. 3B) that possess the CSC properties, i.e.,to generate a new heterogeneous tumor in vivo and tumorspherein vitro (Figs. 3C and 4).
The ALDEFLUOR assay used to isolate CSCs in liquid andsolid tumors detects the activity of several isoforms of ALDH(40). Among them, the main isoforms expressed in tumors areretinaldehyde dehydrogenases, ALDH1A1 and ALDH1A3,responsible for the oxidation of retinal to retinoic acid and,to a lesser extent, ALDH3A1 (20, 40). They can metabolize anddetoxify chemotherapeutic agents such as cyclophosphamidein hematopoietic stem cells, and their level of expression wasshown to be predictive of response to treatment in breast cancer(41, 42). In this study, we show that ALDHþ cells were moreresistant to treatment with conventional chemotherapeuticdrugs than ALDH� cells. We adapted the detection of ALDHactivity with the ALDEFLUOR reagent to fluorescent microsco-py on live cells. Using both methods, we showed that theseALDHþ cells did not incorporate the vital DNA dye Hoechst-33342 instead the ALDH� cells incorporated it, confirming thatALDHþ cells may correspond to the SP of cells with CSCsproperties as previously described by Fukuda and colleaguesin gastric carcinoma cell lines (28). The ability of ALDHþ cellsto efflux Hoechst-33342 and to resist conventional chemother-apy was reversed by verapamil or reserpine treatment, twoinhibitors of efflux pumps such as the ATP-binding cassette(ABC) transporters family members, confirming that these cellsare associated with chemotherapy resistance as proposed inother cancers (43). In this study, we did not find a noticeablecoexpression of BCRP (ABCG2) and MDR-1 (not expressed),the two leaders of the ABC transporters family, in ALDHþ
gastric CSCs (flow cytometry and qRT-PCR analyses, data notshown), suggesting that the Hoechst-33342 and drug effluxmay result from the activity of other members of the ABCtransporters family. This family includes at least 49 genesgrouped into 7 families, and at least 16 of these proteins havebeen implicated in cancer drug resistance (44).
A limit to the use of ALDH as a biomarker of chemoresistantgastric CSC is that ALDH activity can be detected only by theALDEFLUOR assay on live cells by flow cytometry or fluorescentmicroscopy analyses. So, its detection as a biomarker of CSCs incurrent practice on patients' specimens may be possible forcirculating cancer cells and liquid cancers such as leukemia butremain elusive for the analysis of solid tumors such as gastriccarcinoma. These findings also imply that ALDH isozymes can beconsidered not only as biomarkers of CSCs but also as putativetargets to inhibit tumor growth and to overcome resistance tocancer therapy.
It is of importance to note that gastric carcinoma PDXsalways remained heterogeneous and composed of tumor cellsubpopulations expressing EPCAM, CD24, CD133, and CD44,similar to the patients' situation, whereas gastric carcinoma celllines were found to be negative for CD133 and either positiveor negative for CD44 and others markers including ALDH.These results strengthen the importance and the necessity tostudy CSC on models as close as possible to the patients'situation, as is the case in this study, and not only on cancercell lines.
Currently, PDXs represent the most pertinent preclinicalmodel to study the capacity of CSCs to give rise to tumor growth,heterogeneity, and sensitivity/resistance to new treatment strate-gies. However, PDXs models present some limitations,
Nguyen et al.
Clin Cancer Res; 2017 Clinical Cancer ResearchOF10
particularly because the contribution of the patient's tumormicroenvironment—including inflammation, CSCs niche withinthe organ of origin, and cross-talk with immune and stromalcells—cannot be taken into account on CSCs plasticity, tumorprogression, andmetastasis. These limits are partly illustrated hereby the low tumor engraftment success of patient's gastric carci-noma samples, being only approximately 20% of engraftmentsuccess, as described by others for other type of cancers, unveilingthe contribution of uncontrolled microenvironment parametersfor cancer propagation in the patient. Nevertheless, there is anurgent unmet need of new, more efficient and better toleratedtherapeutic strategies for gastric carcinomas, which could focus ongastric CSCs.
In this study, the development of original PDXs modelsallowed us to demonstrate that tumorigenic and chemoresistantgastric CSCs coexpress EPCAM, CD133, CD166, CD44, andALDH, ALDH activity being the most specific biomarker of CSCenrichment before CD44 in both diffuse- and intestinal-typenoncardia gastric carcinomas. This finding led to the hypothesisthat treatment strategy for noncardia gastric carcinomas can focuson CD44þALDHþ CSCs, independently of the histologic classi-fication of the tumor.
Disclosure of Potential Conflicts of InterestL. Wittkop is a consultant/advisory board member for Bristol-Myers Squibb.
No potential conflicts of interest were disclosed by the other authors.
Authors' ContributionsConception and design: P. Dubus, F. Mazurier, C. VaronDevelopment of methodology: P.H. Nguyen, P. Dubus, C. VaronAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Giraud, P. Dubus, G. Belleann�ee, D. Collet,I. Soubeyran, S. Evrard, B. Rousseau, F. Mazurier, C. Varon
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P.H. Nguyen, J. Giraud, P. Dubus, L. Wittkop,C. VaronWriting, review, and/or revision of the manuscript: J. Giraud, P. Dubus,L. Wittkop, F. M�egraud, C. VaronAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L. Chambonnier, N. Senant-DugotStudy supervision: S. Evrard, F. M�egraud, C. Varon
AcknowledgmentsWe thankMarie-Edith Lafon (CNRSUMR5234, University of Bordeaux) and
technicians from theDepartment of Tumor Pathology (Haut-LevequeHospital,University Hospital Center of Bordeaux) for molecular analyses on tumortissues, Pierre Costet (animal facilities, University of Bordeaux), Vincent Pitardand Santiago Gonzalez (Flow Cytometry and FACS Platform, University ofBordeaux), Philippe Brunet de la Grange (CNRS UMR5164 CIRID, Universityof Bordeaux) for assistance on SP cells analyses, and Alban Giese (ExperimentalPathology Platform of the Canceropole GSO and SIRIC BRIO, University ofBordeaux), Elodie Siffre, and Lucie Benejat (INSERM U853) for technicalassistance.
Grant SupportThis study was financially supported by the French "Association pour la
Recherche contre le Cancer" (grant number 8412), the "Institut Nationaldu Cancer" (grant 07/3D1616/IABC-23-12/NC-NG and grant 2014-152),the "Conseil Regional d'Aquitaine" (grant numbers 20071301017 and20081302203), the French National Society for Gastroenterology, and theCanceropole Grand Sud-Ouest (grant 2010-08-canceropole GSO-Universit�eBordeaux 2). This project was also supported by SIRIC BRIO (Site deRecherche Int�egr�ee sur le Cancer – Bordeaux Recherche Int�egr�ee Oncologie;grant INCa-DGOS-Inserm 6046).
The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.
Received September 9, 2015; revised July 19, 2016; accepted July 31, 2016;published OnlineFirst September 12, 2016.
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